Lin28/let-7 crystal structures, purification protocols, and molecular probes suitable for screening assays and therapeutics

ABSTRACT

The invention provides compositions and methods for regulating microRNA (miRNA) biogenesis. The invention also relates to compositions and methods for treating or preventing cancer in a subject in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. Ser. No.15/984,109, filed May 18, 2018, which is a Continuation of U.S. Ser. No.15/420,571 filed Jan. 31, 2017, now a U.S. Pat. No. 10,000,756 issuedJun. 19, 2018, which is a Continuation Application of U.S. Ser. No.14/357,020 filed May 8, 2014, now U.S. Pat. No. 9,593,141 issued Mar.14, 2017, which is a 35 U.S.C. § 371 National Phase Entry Application ofInternational Application No. PCT/US2012/064412 filed Nov. 9, 2012,which designates the U.S., and which claims benefit under 35 U.S.C. §119(e) of the U.S. Provisional Application No. 61/557,655, filed Nov. 9,2011, the contents of which are incorporated herein by reference intheir entireties.

GOVERNMENT SUPPORT

This invention was made with government support under grant no.5U54GM094608 awarded by National Institutes of Health. The governmenthas certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jan. 27, 2017, isnamed 28672131.txt and is 69,026 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to compositions and methods to regulatemicroRNA (miRNA) biogenesis. The invention also relates to compositionsand methods for treating or preventing cancer in a subject in needthereof.

REFERENCES TO TABLES

This application includes as part of the originally filed subject matterthree (3) compact discs, labeled “Copy 1” “Copy 2” and “Copy 3”containing three (3) text files for three (3) separate lengthy tables,which are named “002806072131_TABLE_Uxt” (1,484 KB, created Nov. 9,2012), “002806072131_TABLE2._txt” (541 KB, created Nov. 9, 2012), and“002806072131_TABLE_3.txt” (573 KB, created Nov. 9, 2012). The machineformat of the compact discs (“Copy 1” “Copy 2” and “Copy 3”) is IBM-PCand the operating system of the compact disc is MS-Windows. The contentof the compact disc labeled “Copy 1” “Copy 2” and “Copy 3” is herebyincorporated by reference herein in its entirety. The informationrecorded on Copy 1 is identical to the information recorded on Copy 2and Copy 3.

LENGTHY TABLES

The specification includes three (3) lengthy Tables; Table 1, Table 2,and Table 3. Lengthy Table 1 is the coordinates and structure factorsfor the structures of Lin28:preE_(M)-let-7 and is provided herein in anelectronic format on a CD, as file “002806072131_TABLE_1.txt”. Table 1discloses SEQ ID NOS 118, 119, 111, 119, 111, 119, 111, 119, 111, 119,111, 119, 111, 112, 112, 112, 112, 112, 112, 113, 113, 113, 113, 113,113, 118, 118, 118, 118, 118, 118, 120, 121, 120, 121, 120, 121, 120,121, 120, 121, 120, 121, 118, 118, 118, 118, 118, and 118, respectively,in order of appearance. Lengthy Table 2 is the coordinates and structurefactors for the structures of Lin28:preE_(M)-let-7f-1 and is providedherein in an electronic format on a CD, as file“002806072131_TABLE_2.txt”. Table 2 discloses SEQ ID NOS 17, 111, 111,112, 112, 113, 113, 17, 17, 117, 115, 117, 115, 17, and 17,respectively, in order of appearance. Lengthy Table 3 is the coordinatesand structure factors for the structures of Lin28:preE_(M)-let-7g and isprovided herein in an electronic format on a CD, as file“002806072131_TABLE_3.txt”. Table 3 discloses SEQ ID NOS 18, 111, 111,112, 112, 113, 113, 18, 18, 114, 115, 116, 115, 18, and 18,respectively, in order of appearance. Table 1, Table 2, and Table 3provided herein in an electronic format on a CD, as files“002806072131_TABLE_1.txt”; “002806072131_TABLE_2.txt”; and“002806072131_TABLE_3.txt” respectively are incorporated herein byreference in their entirety. Please refer to the end of thespecification for access instructions.

BACKGROUND

Since the discovery of the first human microRNAs (miRNAs) about a decadeago, examples of miRNA regulation have been found for virtually everycellular process (Kim et al., 2009, Krol et al., 2010). Precursors ofmiRNAs undergo a series of processing steps after transcription togenerate an active product. In this canonical pathway, a newlytranscribed primary miRNA (pri-miRNA) with at least one hairpinstructure is cleaved within the nucleus by an RNAseIII enzyme, Drosha,that acts in complex with DGCR8. The resulting pre-miRNA is exported tothe cytoplasm, where another RNAseIII, Dicer, removes the “terminal loopregion”, or pre-element (preE), to yield the mature miRNA (FIG. 1A).Mechanisms of transcriptional control have been analyzed for manymiRNAs, but the recent identification of post-transcriptional regulatorsof miRNA biogenesis now provides a way to investigate the moleculardetails of miRNA maturation and regulation (Davis-Dusenbery and Hata,2010,Siomi and Siomi, 2010).

The let-7 family of miRNAs regulates many factors that control cell fatedecisions, including oncogenes (c-Myc, Ras, HMGA-2) and cell cyclefactors (CyclinD1, D2) (Bussing et al., 2008,Viswanathan and Daley,2010). Deregulation of let-7 influences tumorigenicity of breast cancerstem cells (Yu et al., 2007a). Moreover, IL-6 is a target of let-7,thereby bridging the inflammation and cell-transformation signalingpathways (Iliopoulos et al., 2009). There are several let-7 familymembers in mammals, with similar mature regions but divergent sequencesin the preE removed by Dicer (FIG. 1A). The preEs comprise low sequenceidentity and thus a minimum motif , i.e., minimal structural elements(stem, bulge, and loop), that are important for regulation of pre-miRNAsare not known (FIG. 1B).

Lin28, originally discovered as a heterochronic gene regulatingdevelopmental timing in worms (Moss et al., 1997), blocks let-7biogenesis (Heo et al., 2008,Lehrbach et al., 2009, Newman et al., 2008,Rybak et al., 2008, Viswanathan et al., 2008). Its effects on geneexpression are profound enough to make Lin28 one of the four factorssufficient to reprogram human somatic cells into induced pluripotentstem (iPS) cells (Yu et al., 2007b). Lin28 is activated in many humantumors (˜15%) and appears to be associated with less differentiatedcancers (Viswanathan et al., 2009). Studies with patient samples showcorrelation between over-expression or mutation of Lin28 with ovariancancer (Peng et al., 2010, Permuth-Wey et al., 2011) and colon cancer(King et al., 2011). Variations in Lin28 have also been linked todevelopmental traits such as height and timing of puberty onset inhumans and mice (Lettre et al., 2008, Lu et al., 2009, Ong et al., 2009,Perry et al., 2009, Sulem et al., 2009, Zhu et al., 2010).

Because it is one of few specific inhibitors of miRNA maturation to bediscovered thus far, understanding Lin28 activity provides an avenue forinvestigating the mechanisms of miRNA biogenesis and regulation. Lin28contains two well-known nucleic acid interaction domains—a cold shockdomain (CSD) and two tandem Cys-Cys-His-Cys (CCHC)-type zinc-bindingmotifs (CCHCx2). Mammals have two paralogs, Lin28a and Lin28b, withdifferent physiological expression patterns but similar behavior invitro (Guo et al., 2006, Heo et al., 2008, Viswanathan et al., 2008,Yang and Moss, 2003). Lin28 binds precursor forms of let-7 miRNAs andcan inhibit both pri-let-7 processing by Drosha (Newman et al., 2008,Viswanathan et al., 2008) and pre-let-7 processing by Dicer (Heo et al.,2008, Lehrbach et al., 2009, Rybak et al., 2008). Furthermore, Lin28 canrecruit a terminal uridylyl transferase (TUTase) that adds uridine tothe 3′ end of pre-miRNA to increase decay (Hagan et al., 2009, Heo etal., 2009, Lehrbach et al., 2009). Although parts of the preE segmentare dispensable for pri-miRNA processing by Drosha (Han et al., 2006),point mutations in the preE can disrupt interactions with Lin28 (Heo etal., 2009, Lehrbach et al., 2009, Newman et al., 2008, Piskounova etal., 2008), thereby de-repressing Drosha-mediated processing (Newman etal., 2008). Sequence variability among preEs in let-7 (FIG. 8A) hashindered interpretation of these results and extension of theconclusions to other let-7s, highlighting the need for an atomic-levelview of divergent Lin28:let-7 complexes.

Accordingly, there is need in the art for inhibitors of Lin28polypeptide activity.

SUMMARY

In one aspect, the invention provides a RNA oligonucleotide or analog,derivative, or pharmaceutically acceptable salt thereof, theoligonucleotide: (a) a nucleotide sequence of formula5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′, wherein N², N⁴, and N⁵ are independently apurine; N⁶ is a pyrimidine; N¹, N³, N⁷, N⁸, and N⁹ are independently anynucleotide; and (b) a nucleotide sequence of 5′-GGAG-3′, wherein thesequence 5′-GGAG-3′ is linked to the 3′ of the sequence of formula5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′, wherein there are from 0 to 100 nucleotidesbetween the 3′ end of 5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′ and 5′ end of thesequence 5′-GGAG-3′, and the sequence 5′-GGAG-3′ is single-stranded.

In some embodiments, there are 0-10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10) nucleotides between the 3′ end of 5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′ and 5′end of the sequence 5′-GGAG-3′, and the sequence 5′-GGAG-3′ issingle-stranded.

In some embodiments, the oligonucleotide comprises a hairpin structurecomprising a hairpin loop and wherein N⁴, N⁵, and N⁶ are in the loopregion of the hairpin.

In another aspect, the invention provides a method for promoting miRNAprocessing of pri-miRNA to mature miRNA in a cell by contacting a cellwith an oligonucleotide described herein.

In yet another aspect, the invention provides a method for treating orpreventing a cancer by administering a therapeutically effective amountto a subject in need thereof.

In still yet another aspect, the invention provides an isolatedpolypeptide comprising amino acids 31-187 of full length Lin28A orLin28B polypeptide, wherein the isolated polypeptide is less than 200amino acids in length. The isolated polypeptide is also referred to asLin28 fragment herein. Lin28 is functional in the presence of two zincatoms which stabilize the two zinc finger domains (CCHCx2) in the Lin28.Accordingly, in some embodiments, two Zn²⁺ atoms are bound with theisolated Lin28 or Lin28B polypeptide.

The invention also provides a crystalline molecule or molecular complexcomprising a binding pocket of Lin28, wherein the Lin28 binding pocketis defined by structure coordinates binding pocket of Table 1, 2, or 3and said Table 1, 2, or 3 being optionally varied by a rmsd of less than1.5 Å or selected coordinates thereof.

Also provided herein is a screening assay for determining inhibitors ofLin28 activity by contacting a test compound with Lin28 fragmentdescribed herein and selecting the compound that increases level ofmature let-7 miRNA relative to a control or inhibits the activity ofLin28 fragment relative to a control.

Provided herein are also method for purification of Lin28 andLin28/Let-7 complexes.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1. Mapping of Lin28 binding sites on pre-let-7. (A) Processingsteps in canonical miRNA biogenesis. Sequences shown are SEQ ID NOS:72-79, 16, and 80-83, respectively, in order of appearance. (B)Architecture of pre-elements (preEs). (C) Fragments of preE-let-7d (SEQID NOS 72-79, 16, and 80-83, respectively, in order of appearance)tested on EMSA for association with Lin28. Affinity is indicated by Kdranges: ++++, 0.2-1.5 μM; +++, 1.5-3 μM; ++, 3-15 μM; +, >15 μM.Predicted stem is highlighted in blue. Minimal fragment (preEM)identified is bolded. See also FIG. 8.

FIG. 2. Linker between CSD and CCHCx2 is flexible. See also FIG. 9. (A)Longitudinal (R1) and transverse (R2) relaxation rates and the ratio(R2/R1), plotted against the residue number. Relatively more dynamicregions are marked with a light yellow box. (B) Alignment of internaldeletions in the linker, indicated with the number of amino acidsdeleted on left (SEQ ID NOS 84-87, respectively, in order ofappearance). (C) EMSAs with preE-let-7d as probe, mixed with increasingconcentrations (0.005, 0.02, 0.08, 0.3, 1.2, 5, 20 μM) of linkerdeletion constructs of Lin28(16-184): *, free probe; **, complex. (D)Quantitative RT-PCR results for in vivo levels of mature let-7g. Lin28Δis truncated at both N and C termini. Lin28ΔΔ has both of the terminalextensions and the linker removed. The standard deviation is calculatedfrom triplicate experiments; U6 RNA levels were used for normalization.(E) and (F) Western blots of Trizol bottom layer for transfections shownin (D). Anti-Lin28 antibodies do not recognize truncation constructs, soanti-FLAG was used in (F) to compare the relative expression levels ofdifferent Lin28 constructs.

FIG. 3. Structure of Lin28:preE_(M)-let-7d complex. Cartoonrepresentations were colored by domain: blue, CSD; green, CCHCx2; grey,zinc; orange, RNA. (A) Stereo representation of the monomeric complex.Interdomain linker is represented by a purple dotted line. (B) Samecomplex in (A) represented with surface colored by electrostaticpotential, and rotated. (C) Domain-swapped dimer. Arrow pointing fromthe domain-swapped to the biologically relevant CCHCx2 domain. Linkerconnecting swapped domains marked in green, dotted line. Linkerconnecting unswapped domains marked in purple, dotted line. See alsoFIG. 10. (D) X-ray data collection and refinement statistics.

FIG. 4. CSD:RNA interactions. See also FIG. 11. (A) Close-up view of CSD(backbone as grey cartoon; key residues also shown with sticks andlabels) interacting with the preE_(M)-loops as labeled, shown in thesame orientation. RNA is colored by base identity (Azure, Ade; Crimson,Cyt; Green, Gua; Umber, Ura), and marked by position number on backboneas defined in text. (B) Schematic drawing of predicted structures ofpreE_(M) sequences (SEQ ID NOS 16-18, respectively, in order ofappearance) used for crystallization. Some of the predicted base pairsare broken (blue vertical dotted line) in the complex structures. Ringsof circles show the protein:RNA interactions at each nucleotideposition, marked with interacting residues (green, hydrophobic orπ-stacking; red, H-bond). Top line is for base contacts and bottom lineis for sugar or phosphate interactions at each position. (C) Comparisonof Lin28 affinity for various preE_(M)-let-7 mutants, described by theparent let-7, position of the mutation, and the base identity.Accompanying gels are shown in FIG. 11. FIG. 4C discloses SEQ ID NOS16-18, respectively, in order of appearance.

FIG. 5. CCHCx2:RNA interactions. See also FIG. 12. (A) Schematic drawingof GGAG, and atoms making contact are marked with amino acid/nucleotidenumbers (green, hydrophobic or 7c-stacking; red, H-bond). (B) Close-upview of base interactions. H-bonds are marked with dashed lines. (C)View of GGAG interactions with CCHCx2. Lin28 is represented with greycartoon, and GGAG are colored by sequence (Green, Gua; Azure, Ade). Zinc(large grey spheres)-coordinating residues are represented with smallspheres at Ca positions (yellow, Cys; cyan, His). (D) Comparison of theCCHCx2 region of Lin28 in different states by superimposition of thefirst CCHC motif (unbound, PDB code=2CQF).

FIG. 6. Structure validation with full-length molecules (A-C) Results ofEMSA with various RNA mutants (full-length pre-miRNA background)combined with protein mutants (full-length Lin28, except for theisolated-domain experiments, designated “CSD or CCHCx2 only”). Affinityindicated by Kd ranges: ++++, 0.13-0.26 μM; +++, 0.26-0.52 μM; ++,0.52-1 μM; +,1-2 μM; −, >2 μM. For isolated domains, scores relative towildtype are given by asterisks, as protein concentrations are higherthan with full-length Lin28. See also FIG. S6. (D) In vitro Dicerprocessing assay with different pre-miR and protein combinations. (E-G)In vivo processing assay as described in FIG. 2D. Full-length Lin28 wasco-transfected with pri-let-7g with indicated mutations (E), or with theentire preE region swapped with preE of an unrelated miR-21 (G)(Piskounova et al., 2008), or pri-miR-122 (G). Wildtype pri-let-7g wasco-transfected with full-length Lin28 constructs with indicatedmutations in (F). Error bars indicate standard deviation calculated fromtriplicate measurements. Immunoblots are shown to compare expressionlevels of Lin28 in each panel.

FIG. 7. Schematic model for Lin28 domains binding to two distinctregions of preE-let-7 (f-1 was used for the model figure). For Lin28:Blue, CSD; Green, CCHCx2; Blue-green loop, protein linker. For pre-let-7depicted as array of cylinders: Yellow, mature region; Orange, basesincluded in the crystallization construct; Grey, parts of preE notincluded in the crystal. Potential partial melting of dsRNA near Dicersites is represented with double-headed arrows; it is uncertain how farthe effect would carry. From structural models, interactions with otherpreE-let-7 sequences can be postulated as shown in FIG. 14.

FIG. 8. Mapping of let-7 precursors for binding to Lin28, Related toFIG. 1. (A) Sequence alignment of pre-elements (preEs) from let-7homologs. Upper block contains mouse homologs, and the lower blockcontains dme (Drosophila melanogaster), crm (Caenorhabditis remanei),cqu (Culex quinquefasciatus), cel (Caenorhabditis elegans), cbr(Caenorhabditis briggasae), and aae (Aedes aegypti). The colors followClustalx scheme. Sequences are SEQ ID NO NOS 88-104, respectively, inorder of appearance. (B) Alignment of the tested oligonucleotidesequences for electrophoretic mobility shift assays. Scores for bindingare defined by Kd ranges: ++++, 0.2-1.5 μM; +++, 1.5-3 μM; ++, 3-15 μM;+, >15 μM. D24 starts with GGAG with the grey sequence attached to the3′ end of the GGAG motif. Sequences are SEQ ID NO NOS 72-79, 16, and80-83, respectively, in order of appearance. (C) Representative EMSAsusing probes indicated, titrated with increasing concentrations ofLin28(16-184) from left to right (8 nM, 33 nM, 130 nM, 520 nM, 2.1 μM,8.3 μM, 33.3 μM). Binding affinities were scored using the major complexband (marked with an asterisk at the right side). Other minor bandssometimes appear, but they are not reproducible and seem to depend onthe protein/RNA batch.

FIG. 9. Domain mapping and structural analysis of Lin28 protein, Relatedto FIG. 2. (A) EMSA of Lin28 truncations. Full-length (1-209) andCSD-CCHC (16-184) constructs have comparable affinity but isolated CSD(16-126) or isolated CCHCx2 (134-184) do not. Full-length preE-let-7dwas used as probe, titrated with protein constructs indicated (left toright: 8 nM, 33 nM, 130 nM, 520 nM, 2.1 μM, 8.3 μM, 33.3 μM). (B)¹⁵N-NOESY of the linker region. Homonuclear 1H/1H NOE spectral stripsbelonging to the residue numbers marked in red were taken from a 3D15N-selected NOESY-HSQC, and only the amide region is shown forinter-residue backbone NOEs. Diagonal peaks are marked with a diagonalline and crosspeaks are marked with a cross. The linker region ismissing inter-residue NOEs, while CCHC has amide-amide interactionsevident from crosspeaks. (C) Plot of secondary shifts for Cα, Cβ, and C′vs. residue number. Domain boundaries, consistent as in FIG. 2A, aremarked with dashed grey lines. (D) EMSA of protein linker deletionconstructs. Increasing amounts of protein (Lin28 35-187, with or without9-residue internal deletion in the linker region) were added toradioactively labeled fragment of pre-let-7f-1 indicated. Theconcentrations of protein in each lane are as follows: 5 nM, 20 nM, 78nM, 313 nM, 1.2 μM, 50 μM, 20 μM. (E) In vivo processing assay ofpri-miR-122 and pri-miR-16, similar to main FIG. 2D. 293T cells (12well) were co-transfected with Lin28 (100 ng) with pri-miR-122 orpri-miR-15-16 (750ng). Standard deviations from three experiments areshown.

FIG. 10. Co-crystallization of Lin28 with sequences from three let-7precursors, Related to FIG. 3. (A) Minimal preE (preE_(M), orange) usedfor co-crystallization is shown for each let-7, in the context of thefull-length preE. Grey nucleotides were removed to reduce flexibilityfor crystallization. For preE-let-7g, another GC pair was added at theposition marked with arrow heads to stabilize the stem structure. Fromleft to rigt, sequences are SEQ ID NO NOS 72, 105 and 106, respectively,in order of appearance. (B) Comparison of Lin28:preE-let-7d structurewith preE-let-7f-1 complex shows that when the CSDs are superimposed,the CCHCx2 shifts according to the longer stem length of preE-let-7f-1.(C) Comparison of Lin28:preE-let-7d structure with preE-let-7g complexshows that due to differences near the ds-ss junction in the CSD bindingregion, the overall axis of the preE-stem is tilted. Again, the CCHCx2follows the GGAG motif, indicating specific binding. (D) Interdomainlinker of preE-let-7g is compared to preE-let-7f-1, and showsvariability. Lack of clear density prevented modeling of the preE-let-7dlinker. (E) Equilibrium sedimentation of WT and truncated linkerconstructs shows that the complexes are monomeric in solution, incontrast to what is seen in co-crystals.

FIG. 11. CSD:RNA interactions, related to FIG. 4. (A) and (B) Stereorepresentation of a detailed view of CSD:preE-loop of the complexindicated. (C) EMSA using the protein and preEM RNA combinationindicated above each row. Each panel represents a titration using theparticular mutation at position marked at the left top corner. Anasterisk indicates a transversion mutation, and plain numbers indicatetransition mutations. Actual sequences for the probes and mutations areshown in main FIG. 4. Sequences are SEQ ID NO NOS 16-18, respectively inorder of appearance. (D) Superimposition of previously determinedCSD:RNA complexes with Lin28:pre-let-7. Only CSD from Lin28 is shownsince all four protein models overlap well (blue cartoon) and RNAbackbone is shown in indicated colors.

FIG. 12. CCHCx2:RNA interactions, related to FIG. 5. (A) Stereorepresentation of a detailed view of CCHCx2:GGAG interactions, usingLin28:preE_(M)-let-7d complex structure. This conformation is observedin all copies of preE-let-7d, both copies of preE-let-7f, and chain B ofpreE-let-7g. (B) Identical orientation as in (A), but protein is shownwith surface representation. (C) Same as (A) but forLin28:preE_(M)-let-7g structure, chain A. (D) Identical orientation as(C), but protein is shown with surface representation. (E) and (F)CCCHCx2 conformations are variable. CCHCx2 from Lin28(green):preE_(M)-let-7d (orange) complex is compared with NMR structuresof HIV NCp1 CCHCx2:RNA complexes by superimposition of the first CCHC.Only RNA backbone is shown in (E) against Lin28, and only proteinbackbone is shown in (F) for clarity.

FIG. 13. Interactions of full-length Lin28 with full-length pre-let-7,related to FIG. 6. (A-C) EMSA using full-length molecules. Gels toaccompany the tables shown in main FIG. 6 are shown here, ordered leftto right and top to bottom, according to the order in each table. (D)Model of the Lin28:pre-let-7 complex binding to Dicer. Dicer and dsRNAsubstrate complex was modeled as referenced in text. The composite modelwith Lin28:preE-let-7 was generated by connecting the preE structure toan ideal dsRNA helix, connected by a flexible pink linker. Due to thelimited number of bases on the 5′ region of preE (hidden behind CCHCx2),Lin28:preE-let-7 crystal structure portion cannot be peeled away fromDicer more without melting much of the mature region (grey loops). Thedirection of CCHCx2 to wedge into the dsRNA is shown with a green arrow.Both CCHCx2 protein volume and unmodeled linker between CSD and CCHCx2would clash with Dicer, as shown with a red arrow. (Blue, CSD; Green,CCHCx2; Orange and Yellow, preE-let-7f-1 included in crystal structure,orange is for direct contacts with Lin28; Pink, preE-let-7f-1 notincluded in the structure; Grey loop, portion of mature region ofpre-let-7; Grey surface, dsRBD and RNAseIIIb dimer from mouse Dicer).

FIG. 14. Structural model suggests how Lin28 would bind to various let-7family members, related to FIG. 7. (A) Proposed Lin28 binding sites on aselection of mouse preE-let-7s. The entire preE-let-7 sequences areshown, as seen in FIG. 8A. The optimal conformation for binding waschosen among the top 3 structures calculated by mfold. The minimal RNAfor CSD binding is shown in red box, with sequence preference(Y=pyrimidine, N=any). The central position in the loop (zero as definedin the text) is marked with an asterisk and the GGAG motif is indicatedwith boxes outlined in black. Although some CSD binding sites do notexactly match the preferred sequence, our mutagenesis results show thatthese RNA mutations only slightly reduce affinity to Lin28. Sequencesshown are SEQ ID NO: 106, for 7a2; SEQ ID NO: 107, for 7b; SEQ ID NO:22, for 7c1; SEQ ID NO: 72, for 7d; SEQ ID NO: 108, for 7e; SEQ ID NO:105, for 7f1; SEQ ID NO: 26, for 7g; and SEQ ID NO: 109, for 7i. (B)Predicted structures of preE-let-7g by mfold as referenced in text.Colored blocks indicate portions used to generate minimal Lin28-bindingconstructs shown in (C). Sequences shown are SEQ ID NO: 26 (left) andSEQ ID NO: 26 (right). (C) Predicted structures of fragments ofpreE-let-7g designed for complex formation with Lin28. Two constructs ofstem-loop with a 3′ tail were designed using the two predictedstructures in (A). An extra G-C base pair was added distal to thepreE-loop to aid with crystallization. EMSA results using the diagrammedprobes in (C) show that conformation 2 construct binds Lin28 with muchhigher affinity. Sequences shown are SEQ ID NO: 110 (left) and SEQ IDNO: 18 (right).

DETAILED DESCRIPTION

The inventors have discovered inter alia that neither the terminal northe linker regions outside of the folded domains of Lin28 polypeptideare essential for blocking let-7 in vivo. The inventors have alsodiscovered that a Lin28 polypeptide fragment comprising truncated N- andC-terminals and deletion in the linker is sufficient for binding topreE-let-7 in vitro. Accordingly, in one aspect, provided herein is anisolated Lin28 polypeptide fragment comprising, consisting or consistingessentially of amino acids 31-187 of a full length Lin28 polypeptide. ALin28 fragment described herein also includes analogs, derivatives, andfunctional conservatives of said Lin28 fragment. As used herein, a Lin28polypeptide encompasses both Lin28A and Lin28B polypeptides.

A Lin28 fragment described herein does not comprise the full lengthLin28 polypeptide sequence. Accordingly, length of a Lin28 fragmentdescribed herein is at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more)amino acid less than the full length of Lin28A or Lin28B polypeptide.Generally, a Lin28 fragment described herein has a deletion of from 1 to30 amino acids from the N-terminal or from 1 to 22 amino acids from theC-terminal of a full length Lin28 polypeptide.

In some embodiments, a Lin28 fragment described herein is less than 200amino acids in length. In some embodiments, the Lin28 fragment is lessthan 175 amino acids in length.

In some embodiments, the Lin28 polypeptide fragment further comprises adeletion in the linker region of a full length Lin28 polypeptide. Asused herein, the linker region of the Lin28 polypeptide refers to theamino acid sequence connecting the CSD and the first CCHC domain in thefull length Lin28 polypeptide. Generally, the linker region is locatedbetween amino acid positions 121 to 138 of the full length Lin28polypeptide. In some embodiments, the Lin28 polypeptide fragmentcomprises a deletion of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,or 18 amino acids from the linker region. In some embodiments, Lin28polypeptide fragment comprises deletion of amino acids 127 to 135 of thefull length of a Lin28 polypeptide. In some embodiments, Lin28polypeptide fragment comprises deletion of amino acids 121 to 135 of thefull length of a Lin28 polypeptide.

In some embodiments, the Lin28 fragment comprises at least one (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) amino acid residue selected from the group consisting ofhomocysteine, phosphoserine, phosphothreonine, phosphotyrosine,hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine(3-mercapto-D-valine), ornithine, citruline, alpha-methyl-alanine,para-benzoylphenylalanine, para-aminophenylalanine,p-fluorophenylalanine, phenylglycine, propargylglycine, sarcosine, andtert-butylglycine), diaminobutyric acid,7-hydroxy-tetrahydroisoquinoline carboxylic acid, naphthylalanine,biphenylalanine, cyclohexylalanine, amino-isobutyric acid, norvaline,norleucine, tert-leucine, tetrahydroisoquinoline carboxylic acid,pipecolic acid, phenylglycine, homophenylalanine, cyclohexylglycine,dehydroleucine, 2,2-diethylglycine, 1-amino-1-cyclopentanecarboxylicacid, 1-amino-1-cyclohexanecarboxylic acid, amino-benzoic acid,amino-naphthoic acid, gamma-aminobutyric acid, difluorophenylalanine,nipecotic acid, alpha-amino butyric acid, thienyl-alanine,t-butylglycine, desamino-Tyr, aminovaleric acid, pyroglutaminic acid,alpha-aminoisobutyric acid, gamma-aminobutyric acid, alpha-aminobutyricacid, alpha,gamma-aminobutyric acid, pyridylalanine, α-napthyalanine,β-napthyalanine, Ac-β-napthyalanine, N^(ε)-picoloyl-lysine,4-halo-Phenyl, 4-pyrolidylalanine, isonipecotic carboxylic acid, and anycombinations thereof.

In some embodiments, the Lin28 fragment comprises at least one (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) D-amino acid. Without limitations, the D-amino acid can be presentat any position in the Lin28 fragment. When more than one D-amino acidsare present, they can be positioned next to or not next to each other.When three or more D-amino acids are present some of the D-amino acidscan be present next to another D-amino acid while some of the D-aminoacids are not next to another D-amino acid

In some embodiments, the Lin28 fragment comprises a chemically modifiedamino acid. Such a chemically modified amino acid can be present at anyposition in the Lin 28 fragment. Additionally, when more than onechemically modified amino acids are present, they can be positioned nextto or not next to each other. When three or more chemically modifiedamino acids are present some of the chemically modified amino acids canbe present next to each other while some of the chemically modifiedamino are not next to another chemically modified amino acid. As usedherein, the term “chemically modified amino acid” refers to an aminoacid that has been treated with one or more reagents.

In some embodiments, the Lin28 fragment comprises at least one (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) beta-amino acid. The beta-amino acid can be present at anyposition in the Lin28 fragment. Further, when more than one beta-aminoacids are present, they can be positioned next to or not next to eachother. When three or more beta-amino acids are present some of thebeta-amino acids can be present next to another beta-amino acid whilesome of the beta-amino are not next to another beta-amino acid.

Exemplary beta-amino acids include, but are not limited to,L-β-Homoproline hydrochloride; (±)-3-(Boc-amino)-4-(4-biphenylyl)butyricacid; (±)-3-(Fmoc-amino)-2-phenylpropionic acid;(1S,3R)-(+)-3-(Boc-amino)cyclopentanecarboxylic acid;(2R,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid;(2S,3R)-3-(Boc-amino)-2-hydroxy-4-phenylbutyric acid;(R)-2-[(Boc-amino)methyl]-3-phenylpropionic acid;(R)-3-(Boc-amino)-2-methylpropionic acid;(R)-3-(Boc-amino)-2-phenylpropionic acid;(R)-3-(Boc-amino)-4-(2-naphthyl)butyric acid;(R)-3-(Boc-amino)-5-phenylpentanoic acid;(R)-3-(Fmoc-amino)-4-(2-naphthyl)butyric acid;(R)-(−)-Pyrrolidine-3-carboxylic acid; (R)-Boc-3,4-dimethoxy-β-Phe-OH;(R)-Boc-3-(3-pyridyl)-β-Ala-OH; (R)-Boc-3-(trifluoromethyl)-β-Phe-OH;(R)-Boc-3-cyano-β-Phe-OH; (R)-Boc-3-methoxy-β-Phe-OH;(R)-Boc-3-methyl-β-Phe-OH; (R)-Boc-4-(4-pyridyl)-β-Homoala-OH;(R)-Boc-4-(trifluoromethyl)-β-Homophe-OH;(R)-Boc-4-(trifluoromethyl)-β-Phe-OH; (R)-Boc-4-bromo-β-Phe-OH;(R)-Boc-4-chloro-β-Homophe-OH; (R)-Boc-4-chloro-β-Phe-OH;(R)-Boc-4-cyano-β-Homophe-OH; (R)-Boc-4-cyano-β-Phe-OH;(R)-Boc-4-fluoro-β-Phe-OH; (R)-Boc-4-methoxy-β-Phe-OH;(R)-Boc-4-methyl-β-Phe-OH; (R)-Boc-β-Tyr-OH;(R)-Fmoc-4-(3-pyridyl)-β-Homoala-OH; (R)-Fmoc-4-fluoro-β-Homophe-OH;(S)-(+)-Pyrrolidine-3-carboxylic acid;(S)-3-(Boc-amino)-2-methylpropionic acid;(S)-3-(Boc-amino)-4-(2-naphthyl)butyric acid;(S)-3-(Boc-amino)-5-phenylpentanoic acid;(S)-3-(Fmoc-amino)-2-methylpropionic acid;(S)-3-(Fmoc-amino)-4-(2-naphthyl)butyric acid;(S)-3-(Fmoc-amino)-5-hexenoic acid;(S)-3-(Fmoc-amino)-5-phenyl-pentanoic acid;(S)-3-(Fmoc-amino)-6-phenyl-5-hexenoic acid;(S)-Boc-2-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-2-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-2-(trifluoromethyl)-β-Phe-OH; (S)-Boc-2-cyano-β-Homophe-OH;(S)-Boc-2-methyl-β-Phe-OH; (S)-Boc-3,4-dimethoxy-β-Phe-OH;(S)-Boc-3-(trifluoromethyl)-β-Homophe-OH;(S)-Boc-3-(trifluoromethyl)-β-Phe-OH; (S)-Boc-3-methoxy-β-Phe-OH;(S)-Boc-3-methyl-β-Phe-OH; (S)-Boc-4-(4-pyridyl)-β-Homoala-OH;(S)-Boc-4-(trifluoromethyl)-β-Phe-OH; (S)-Boc-4-bromo-β-Phe-OH;(S)-Boc-4-chloro-β-Homophe-OH; (S)-Boc-4-chloro-β-Phe-OH;(S)-Boc-4-cyano-β-Homophe-OH; (S)-Boc-4-cyano-β-Phe-OH;(S)-Boc-4-fluoro-β-Phe-OH; (S)-Boc-4-iodo-β-Homophe-OH;(S)-Boc-4-methyl-β-Homophe-OH; (S)-Boc-4-methyl-β-Phe-OH;(S)-Boc-β-Tyr-OH; (S)-Boc-y,y-diphenyl-β-Homoala-OH;(S)-Fmoc-2-methyl-β-Homophe-OH; (S)-Fmoc-3,4-difluoro-β-Homophe-OH;(S)-Fmoc-3-(trifluoromethyl)-β-Homophe-OH;(S)-Fmoc-3-cyano-β-Homophe-OH; (S)-Fmoc-3-methyl-β-Homophe-OH;(S)-Fmoc-y,y-diphenyl-β-Homoala-OH; 2-(Boc-aminomethyl)phenylaceticacid; 3-Amino-3-(3-bromophenyl)propionic acid;3-Amino-4,4,4-trifluorobutyric acid; 3-Aminobutanoic acid;DL-3-Aminoisobutyric acid; DL-β-Aminoisobutyric acid puriss;DL-β-Homoleucine; DL-β-Homomethionine; DL-β-Homophenylalanine;DL-I3-Leucine; DL-β-Phenylalanine; L-β-Homoalanine hydrochloride;L-β-Homoglutamic acid hydrochloride; L-β-Homoglutamine hydrochloride;L-β-Homohydroxyproline hydrochloride; L-β-Homoisoleucine hydrochloride;L-β-Homoleucine hydrochloride; L-β-Homolysine dihydrochloride;L-β-Homomethionine hydrochloride; L-β-Homophenylalanine allyl esterhydrochloride; L-β-Homophenylalanine hydrochloride; L-β-Homoserine;L-β-Homothreonine; L-β-Homotryptophan hydrochloride; L-β-Homotyrosinehydrochloride; L-β-Leucine hydrochloride; Boc-D-β-Leu-OH;Boc-D-β-Phe-OH; Boc-β³-Homopro-OH; Boc-β-Glu(OBzl)-OH;Boc-β-Homoarg(Tos)-OH; Boc-β-Homoglu(OBzl)-OH; Boc-β-Homohyp(Bzl)-OH(dicyclohexylammonium) salt technical; Boc-β-Homolys(Z)-OH;Boc-β-Homoser(Bzl)-OH; Boc-13-Homothr(Bzl)-OH; Boc-β-Homotyr(Bzl)-OH;Boc-β-Ala-OH; Boc-β-Gln-OH; Boc-β-Homoala-OAll; Boc-β-Homoala-OH;Boc-β-Homogln-OH; Boc-β-Homoile-OH; Boc-β-Homoleu-OH; Boc-β-Homomet-OH;Boc-β-Homophe-OH; Boc-β-Homotrp-OH; Boc-β-Homotrp-OMe; Boc-β-Leu-OH;Boc-β-Lys(Z)-OH (dicyclohexylammonium) salt; Boc-β-Phe-OH; Ethyl3-(benzylamino)propionate; Fmoc-D-β-Homophe-OH; Fmoc-L-β³-homoproline;Fmoc-β-D-Phe-OH; Fmoc-β-Gln(Trt)-OH; Fmoc-β-Glu(OtBu)-OH;Fmoc-β-Homoarg(Pmc)-OH; Fmoc-β-Homogln(Trt)-OH; Fmoc-β-Homoglu(OtBu)-OH;Fmoc-β-Homohyp(tBu)-OH; Fmoc-β-Homolys(Boc)-OH; Fmoc-β-Homoser(tBu)-OH;Fmoc-β-Homothr(tBu)-OH; Fmoc-β-Homotyr(tBu)-OH; Fmoc-β-Ala-OH;Fmoc-β-Gln-OH; Fmoc-β-Homoala-OH; Fmoc-β-Homogln-OH; Fmoc-β-Homoile-OH;Fmoc-β-Homoleu-OH; Fmoc-β-Homomet-OH; Fmoc-β-Homophe-OH;Fmoc-β-Homotrp-OH; Fmoc-β-Leu-OH; Fmoc-β-Phe-OH;N-Acetyl-DL-β-phenylalanine; Z-D-β-Dab(Boc)-OH; Z-D-β-Dab(Fmoc)-OHpurum; Z-DL-β-Homoalanine; Z-β-D-Homoala-OH; Z-β-Glu(OtBu)-OH technical;Z-β-Homotrp(Boc)-OH; Z-β-Ala-OH purum; Z-β-Dab(Boc)-OH;Z-β-Dab(Fmoc)-OH; Z-β-Homoala-OH; β-Alanine; β-Alanine BioXtra;β-Alanine ethyl ester hydrochloride; β-Alanine methyl esterhydrochloride; β-Glutamic acid hydrochloride;cis-2-Amino-3-cyclopentene-1-carboxylic acid hydrochloride;cis-3-(Boc-amino)cyclohexanecarboxylic acid; andcis-3-(Fmoc-amino)cyclohexanecarboxylic acid.

In some embodiments, the Lin28 fragment comprises at least one (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, ormore) modified peptide linkage, e.g., a peptide bond replaced by alinkage selected from the group consisting of reduced psi peptide bond,urea, thiourea, carbamate, sulfonyl urea, trifluoroethylamine,ortho-(aminoalkyl)-phenylacetic acid, para-(aminoalkyl)-phenylaceticacid, meta-(aminoalkyl)-phenylacetic acid, thioamide, tetrazole, boronicester, and olefinic group. The peptide replacement linkage can bepresent at any position in the Lin2 fragment. When more than peptidereplacement linkages are present, they can be positioned next to (e.g.,on both sides of a given amino acid) or not next to each other (e.g.,only one side of a given amino acid is linked via a peptide replacementlinkage to the next amino acid).

In some embodiments, the N-terminus amino group of the Lin28 peptideconjugated with nitrogen- or amino-protecting group. As used herein, a“nitrogen protecting group” or an “amino protecting group” refers tomoieties that block or mask the NH2 group. Exemplary amino-protectinggroups include, but are not limited to, carbamate protecting groups,such as 2-trimethylsilylethoxycarbonyl (Teoc),1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl (BOC),allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc), andbenzyloxycarbonyl (Cbz); amide protecting groups, such as formyl,acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl; sulfonamideprotecting groups, such as 2-nitrobenzenesulfonyl; and imine and cyclicimide protecting groups, such as phthalimido and dithiasuccinoyl.Further amino protecting groups, as well as other representativeprotecting groups, are disclosed in Greene and Wuts, Protective Groupsin Organic Synthesis, Chapter 2, 2d ed., John Wiley & Sons, New York,1991, and Oligonucleotides And Analogues A Practical Approach, Ekstein,F. Ed., IRL Press, N.Y, 1991, content of which is herein incorporated byreference in its entirety.

In some embodiments, the N-terminus amino acid of the Lin28 fragment isacetylated or alkylated, e.g., with acetyl, ethanoyl, propionyl,t-butanoyl, methyl, ethyl, propyl, butyl, pentyl, or hexanyl.

In some embodiments, the N-terminus amino acid of the Lin28 fragment isconjugated with a tag amino acid sequence. Without wishing to be boundby a theory, a tag sequence makes it easy to synthesize and purify thepolypeptide. In one embodiment, the tag amino acid sequence isMHHHHHHENLYFQ (SEQ ID NO: 1).

In some embodiments, the Lin28 fragment is conjugated with polyethyleneglycol (PEG). Without wishing to be bound by theory, such conjugationcan increase the in vivo half-life of the Lin28 fragment. As usedherein, “PEG” means an ethylene glycol polymer that contains about 20 toabout 2000000 linked monomers, typically about 50-1000 linked monomers,usually about 100-300. Polyethylene glycols include PEGs containingvarious numbers of linked monomers, e.g., PEG20, PEG30, PEG40, PEG60,PEG80, PEG100, PEG115, PEG200, PEG 300, PEG400, PEG500, PEG600, PEG1000,PEG1500, PEG2000, PEG3350, PEG4000, PEG4600, PEG5000, PEG6000, PEG8000,PEG11000, PEG12000, PEG2000000 and any mixtures thereof. Methods ofconjugating PEGs to peptides are well known in the art. A Lin28fragmentcan comprise a PEG at the N-terminus, C-terminus, or at an internalamino acid. The PEG can be linked to the N-terminus amino group,C-terminus carboxyl group, or to an amino, hydroxyl or thiol group onthe side chain of an amino acid.

As Lin28 is functional in the presence of two Zinc atoms which stabilizetwo zinc finger domains (CCHCx2), in some embodiments, two divalentcations can be bound to the isolated Lin28 or Lin28B polypeptidedescribed herein. As used in this application, the term “divalentcation” means a cation having a +2 charge. Exemplary divalent cationsinclude, but are not limited to, zinc, cobalt, nickel, cadmium,magnesium, and manganese. In some embodiments, the divalent cation isZn²⁺.

In some embodiments, the Lin28 fragment is selected from the groupconsisting of:

(SEQ ID NO: 2) MHHHHHHENLYFQGSGAAEKAPEEAPPDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGKNMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSINHMVASCPLKAQQGPSS(mouse Lin28A delta + tag); (SEQ ID NO: 3)MHHHHHHENLYFQGSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHHAKECKLPPQPKKCHFC QSINHMVASCPLKAQQGPSSQGK(mouse Lin28A delta delta + tag); (SEQ ID NO: 4)MHHHHHHENLYFQGSGEEPEKLPGLAEDEPQVLHGTGHCKWFNVRMGFGFISMISREGNPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSPKGLESIRVTGPGGSPCLGSERRPKGKTLQKRKPKGDRWRRQDLLMDQMWTVREEESRMIPRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMH MVANCPHKLAAQLPASS(mouse Lin28B delta + tag); (SEQ ID NO: 5)MHHHHHHENLYFQGSGAAEEAPEEAPEDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGKSMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLKAQQGPSAQGK(human Lin28A delta + tag); (SEQ ID NO: 6)MHHHHHHENLYFQGSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHHAKECKLPPQPKKCHFC QSISHMVASCPLKAQQGPSAQGK(human Lin28A delta delta + tag), (SEQ ID NO: 7)MHHHHHHENLYFQGSGEEPGKLPEPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGSERRPKGKTLQKRKPKGDRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKNVAQPPASSQGR (human Lin28B delta + tag),(SEQ ID NO: 8) MHHHHHHENLYFQGSGPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGSERRPKGGDRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKNVAQPPASSQGR (human Lin28B delta delta + tag),(SEQ ID NO: 9) GSGAAEKAPEEAPPDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGKNMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSINHMVASCPLKAQQGPSS (mouse Lin28A delta), (SEQ ID NO: 10)GSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSINHMVASCPLK AQQGPSSQGK(mouse Lin28A delta delta), (SEQ ID NO: 11)GSGEEPEKLPGLAEDEPQVLHGTGHCKWFNVRMGFGFISMISREGNPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSPKGLESIRVTGPGGSPCLGSERRPKGKTLQKRKPKGDRWRRQDLLMDQMWTVREEESRMIPRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKLAAQL PASS (mouse Lin28B delta), (SEQ ID NO: 12)GSGAAEEAPEEAPEDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGKSMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLKAQQGPSAQGK (human Lin28A delta), (SEQ ID NO: 13)GSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLK AQQGPSAQGK (human Lin28A delta delta), (SEQ ID NO: 14)GSGEEPGKLPEPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGSERRPKGKTLQKRKPKGDRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKNVAQPPASSQGR (human Lin28B delta), and (SEQ ID NO: 15)GSGPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVT (human Lin28B delta delta).

A Lin28 fragment described herein can be synthesized according to theusual methods of solution and solid phase peptide chemistry, or byclassical methods known in the art. Purification of peptides is wellknown in the art and can be, for example, HPLC. Methods describinguseful peptide synthesis and purification methods can be found, forexample, in U.S. Patent Application No. 20060084607.

Lin28 fragments described herein can be synthetically constructed bysuitable known peptide polymerization techniques, such as exclusivelysolid phase techniques, partial solid-phase techniques, fragmentcondensation or classical solution couplings. For example, the peptidesof the invention can be synthesized by the solid phase method usingstandard methods based on either t-butyloxycarbonyl (BOC) or9-fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodologyis described by G. B. Fields et al. in Synthetic Peptides: A User'sGuide, W. M. Freeman & Company, New York, N.Y., pp. 77-183 (1992) and inthe textbook “Solid-Phase Synthesis”, Stewart & Young, Freemen &Company, San Francisco, 1969, and are exemplified by the disclosure ofU.S. Pat. No. 4,105,603, issued Aug. 8, 1979. Classical solutionsynthesis is described in detail in “Methoden der Organischen Chemic(Houben-Weyl): Synthese von Peptiden”, E. Wunsch (editor) (1974) GeorgThieme Verlag, Stuttgart West Germany. The fragment condensation methodof synthesis is exemplified in U.S. Pat. No. 3,972,859. Other availablesyntheses are exemplified in U.S., Pat. Nos. 3,842,067 and 3,872,925,Merrifield B, Protein Science (1996), 5: 1947-1951; The chemicalsynthesis of proteins; Mutter M, Int J Pept Protein Res 1979 March; 13(3): 274-7 Studies on the coupling rates in liquid-phase peptidesynthesis using competition experiments; and Solid Phase PeptideSynthesis in the series Methods in Enzymology (Fields, G. B. (1997)Solid-Phase Peptide Synthesis. Academic Press, San Diego, #9830).Contents of all of the foregoing disclosures are incorporated herein byreference.

In some embodiments, Lin28 constructs, e.g., Lin28 constrcuts derivedfrom mouse Lin28a, can be purified after overexpression in E.coli, usingNickel affinity, cation exchange, and size exclusion chromatography. Forexample, Lin28 constructs can be overexpressed in E. coli strainBL21(DE3) Rosetta pLysS. After initial affinity chromatography stepusing Ni-NTA beads (Qiagen), His-tags can be removed by incubating withrecombinant TEV protease. After His-tag removal, the Lin28 constrcutscan be purified by cation exchange chromatography. Cation exchangechromatography can be performed using a HiTrap S (GE Healthcare) with abuffer containing 20 mM BisTris pH 6.0, 5 mM dithiothreitol (DTT), 5%glycerol, and 50 μM ZnCl₂, over 0.1-1M NaCl gradient. If desired,further purification of the Lin28 constructs can be accomplished by sizeexclusion chromatography. For example, size exclusion chromatography canbe performed using Superdex 200 (GE Healthcare) in the same buffer asnoted-above. In some embodiments, a Lin28 functional fragment can besynthesized and purified as described herein in Example 1.

Without wishing to be bound by a theory, a Lin28 fragment describedherein can be used in place of the full length Lin28 polypeptide inmethods requiring the use of a full length Lin28 polypeptide. Asdescribed herein, a Lin28 fragment can bind preE-let-7 both in vivo andin vitro. Thus, a Lin28 fragment described herein has similar preE-let-7binding activity as the full length Lin28 polypeptide.

One method of producing induced pluripotent stem cells, comprisesintroducing a Lin28 polypeptide into a cell. Because a Lin28 fragmenthas the similar activity as the full length polypeptide, a Lin28fragment of the invention can be used in methods of producing inducedpluripotent stem cells. Exemplary methods of producing inducedpluripotent stem cells using Lin28 are described for example in U.S.Pat. App. Pub. No. 2011/0117653, No.2011/0200568, No. 2011/044961, No.2011/0039338, No. 2010/0062533, No. 2011/0250692, No. 2011/0003365, No.2011/0236966, No. 2011/0201110, No. 2011/0244566, No. 2011/0104805, No.2011/0143436, No. 2011/0190729, No. 2010/0041054, No. 2010/0093092, andNo. 2010/0120069, content of all of which is incorporated herein byreference. Without wishing to be bound by a theory, a Lin28 fragmentdisclosed herein can be used in place of a full length Lin28 polypeptidein the methods described in the above-noted references.

A Lin28 fragment described herein is sufficient for binding topreE-let-7 in vitro. Accordingly, the Lin28 fragment can be used forscreening inhibitors of full length Lin28 polypeptide or agents thatpromote miRNA processing of pre-miRNA to mature miRNA. Accordingly,provided herein is a method for screening a test compound for inhibitingactivity of Lin28 polypeptide or promoting processing of pre-miRNA tomature miRNA, the method comprising contacting a Lin28 fragmentdescribed herein with a test compound.

Alternatively, a test compound can be assessed for its ability tofunction as an inhibitor of Lin28 polypeptide by assessing the cellproliferation (or cell growth) of a cancer cell line, such as the H1299lung adenocarcinoma or chronic myelogenous leukemia (CML) cell lines (asdisclosed herein in the Examples) in the presence of the test compoundinhibitor of Lin28. An decrease in the cell proliferation rate (or cellgrowth) in the presence of the inhibitor of Lin28 as compared to in theabsence of an agent, or a negative control indicates the test compoundfunctions as an inhibitor of Lin28. Alternatively, a substantiallysimilar i.e. about at least 60%, or at least about 70%, or at leastabout 80% or at least about 90% or more cell proliferation rate (or cellgrowth) in the presence of the test compound inhibitor of Lin28 ascompared to the presence of a positive control (i.e. an oligonucleotidedescribed herein) indicates the test compound functions as an inhibitorof Lin28.

In some embodiments, the method further comprises selecting the compoundthat promotes processing of a pre-miRNA to a mature miRNA or thatinhibits binding of oligonucleotide described herein, a pre-miRNA, ormiRNA to the Lin28 fragment.

For the screening assays, the Lin28 fragment can be immobilized on asolid support. Polypeptides can be immobilized using methods known inthe art, such as adsorption onto a plastic microtiter plate or specificbinding of a GST-fusion protein to a polymeric bead containingglutathione. For example, a GST-Lin28 fragment can be bound toglutathione-Sepharose beads. The immobilized peptide is then contactedwith the test compound.

As used herein, the term “test compound” refers to compounds and/orcompositions that are to be screened for their ability to inhibit Lin28activity or to promote miRNA processing. Test compounds may include awide variety of different compounds, including chemical compounds,mixtures of chemical compounds, e.g., small organic or inorganicmolecules; saccharides; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., peptides, proteins, and peptide analogs andderivatives; peptidomimetics; nucleic acids; nucleic acid analogs andderivatives; an extract made from biological materials such as bacteria,plants, fungi, or animal cells; animal tissues; naturally occurring orsynthetic compositions; and any combinations thereof. In someembodiments, the test compound is a small molecule.

As used herein, the term “small molecule” can refer to compounds thatare “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kD), preferablyless than 3 kD, still more preferably less than 2 kD, and mostpreferably less than 1 kD. In some cases it is preferred that a smallmolecule have a molecular mass equal to or less than 700 Daltons.

The number of possible test compounds runs into millions. Methods fordeveloping small molecule, polymeric and genome based libraries aredescribed, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596(2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001).Commercially available compound libraries can be obtained from, e.g.,ArQule, Pharmacopia, Graffinity, Panvera, Vitas-M Lab, BiomolInternational and Oxford. Other known compound libraries include, NIHClinical Collection 1 and 2, Biomol 4—FDA Approved Drugs, Sigma Lopac,Tocriscreen Mini Library, NINDS Custom Collection 2, Prestwick 2Collection, MSDiscovery 1, Biomol ICCB Know Bioactives, Asinex 1,ChemBridge 3, ChemDiv 4 and 6, Life Chemicals 1, and Maybridge 4 and 5.These libraries can be screened using the screening devices and methodsdescribed herein. Chemical compound libraries such as those from NIHRoadmap, Molecular Libraries Screening Centers Network (MLSCN) can alsobe used. A comprehensive list of compound libraries can be found on theweb atwww.broad.harvard.edu/chembio/platform/screening/compound_libraries/index.htm.

A chemical library or compound library is a collection of storedchemicals usually used ultimately in high-throughput screening orindustrial manufacture. The chemical library can consist in simple termsof a series of stored chemicals. Each chemical has associatedinformation stored in some kind of database with information such as thechemical structure, purity, quantity, and physiochemical characteristicsof the compound.

Depending upon the particular embodiment being practiced, the testcompounds can be provided free in solution, or may be attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports may be employed for immobilization of the test compounds.Examples of suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods described herein, test compounds may bescreened individually, or in groups. Group screening is particularlyuseful where hit rates for effective test compounds are expected to below such that one would not expect more than one positive result for agiven group.

Generally, compounds can be tested at any concentration. In someembodiments, compounds are tested at a concentration in the range offrom about 0.1 nM to about 1000 mM. Preferably the compound is tested inthe range of from about 0.1μM to about 10μM.

Additionally, the test compound can be contacted with the Lin28 fragmentfor a sufficient time to allow the test compound to interact with theLin28 fragment. For a non-limiting example, Lin28 fragment is incubatedwith the test compound for at least 15 minutes before assaying foractivity.

In some embodiments, screening assay further comprises selecting thecompound that inhibits or reduces Lin28 activity. The test compound caninhibit Lin28 activity by at least 10%, 20%, 30%, 40%, 50%, 50%, 70%,80%, 90%, 95% or more relative to a control.

In some embodiments, the screening method is a high-throughputscreening. High-throughput screening (HTS) is a method for scientificexperimentation that uses robotics, data processing and controlsoftware, liquid handling devices, and sensitive detectors.High-Throughput Screening or HTS allows a researcher to quickly conductmillions of biochemical, genetic or pharmacological tests.High-Throughput Screening methods are well known to one skilled in theart, for example, those described in U.S. Pat. Nos. 5,976,813;6,472,144; 6,692,856; 6,824,982; and 7,091,048, and contents of each ofwhich is herein incorporated by reference in its entirety.

HTS uses automation to run a screen of an assay against a library ofcandidate compounds. An assay is a test for specific activity: usuallyinhibition or stimulation of a biochemical or biological mechanism.Typical HTS screening libraries or “decks” can contain from 100,000 tomore than 2,000,000 compounds.

The key labware or testing vessel of HTS is the microtiter plate: asmall container, usually disposable and made of plastic that features agrid of small, open divots called wells. Modern microplates for HTSgenerally have either 384, 1536, or 3456 wells. These are all multiplesof 96, reflecting the original 96 well microplate with 8×12 9 mm spacedwells.

To prepare for an assay, the researcher fills each well of the platewith the appropriate reagents that he or she wishes to conduct theexperiment with, such as Ephexin5. After some incubation time has passedto allow the reagent to absorb, bind to, or otherwise react (or fail toreact) with the compounds in the wells, measurements are taken acrossall the plate's wells, either manually or by a machine. Manualmeasurements are often necessary when the researcher is using microscopyto (for example) seek changes that a computer could not easily determineby itself. Otherwise, a specialized automated analysis machine can run anumber of experiments on the wells such as colorimetric measurements,radioactivity counting, etc. In this case, the machine outputs theresult of each experiment as a grid of numeric values, with each numbermapping to the value obtained from a single well. A high-capacityanalysis machine can measure dozens of plates in the space of a fewminutes like this, generating thousands of experimental data points veryquickly.

In another aspect, the invention provides a compound selected by thescreening assay described herein. It is to be understood that analogs,derivatives, and isomers of the compounds selected by the screeningassays described herein are also claimed herein.

The structural information disclosed herein is useful analysis ofbinding interactions with a ligand, e.g., for discovery of inhibitors ofLin28 polypeptide activity. Such data is useful for a number ofpurposes, including the generation of structures to analyze themechanisms of action and/or to discover or perform rational drug designof active compounds. For example, a search of several small-moleculestructural data bases such as Available Chemicals Directory, CambridgeCrystallographic Database, Fine Chemical Database and CONCORD databaseis carried out using parameters derived from the crystal structuresdescribed herein. The search can be 2-dimensional, 3-dimensional or bothand can be done using a combination of software such as UNITY version2.3.1 (Tripos, Inc.), MACCS 3D, CAVEAT and DOCK. Conformationalflexibility of the small molecules is allowed. The strategy forconducting the search takes into account conformations and/or keyresidues in the combining site

Structural information disclosed herein can be stored on acomputer-readable medium. The invention therefore provides systems,particularly a computer system, the systems containing the atomicco-ordinate data of any one of the tables below, or selectedco-ordinates thereof The computer system can comprise: (i) acomputer-readable data storage medium comprising data storage materialencoded with the computer-readable data; (ii) a working memory forstoring instructions for processing said computer-readable data; and(iii) a central-processing unit coupled to said working memory and tothe computer-readable data storage medium for processing saidcomputer-readable data and thereby generating structures and/orperforming rational drug design. The computer system can furthercomprise a display coupled to the central-processing unit for displayingsaid structures. The computer system can contain one or more remotedevices. The remote device may comprise e.g. a computer system orcomputer readable media of one of the previous aspects of the invention.The device can be in a different country or jurisdiction from where thecomputer-readable data is received. The communication with a remotedevice may be via the internet, intranet, and e-mail etc. . . . ,transmitted through wires or by wireless means such as by terrestrialradio or by satellite. Typically the communication will be electronic innature, but some, or all, of the communication pathway may be optical,for example, over optical fibers. The data received can then be used ina computer-based method for the analysis of the interaction of a ligandas discussed above.

Based on the elucidated structures of Lin28:preE-let-7 complexes, theinventors have discovered the minimum motif for an RNA oligonucleotidethat can bind to a Lin28 polypeptide. The inventors have discovered thatthe sequence 5′-N1N2N3N4N5N6N7N8N9-3′ provides the minimum domaincapable of binding the CSD domain of Lin28 and the sequence 5′-GGAG-3′is sufficient for binding to the CCHC domain of Lin28. Further a linkerof zero or more nucleotides in between the two sequences is sufficientto allow an oligonucleotide to bind with the Lin28.

Accordingly, provided herein is an RNA oligonucleotide comprising atleast two different domains for binding to a Lin28 polypeptide. Theoligonucleotide comprises: (a) a nucleotide sequence of formula5′-N1N2N3N4N5N6N7N8N9-3′, wherein N2, N4, and N5 are independently apurine; N6 is a pyrimidine; N1, N3, N7, N8, and N9 are independently anynucleotide; and (b) a single-stranded nucleotide sequence of 5′-GGAG-3′,wherein the two sequences are linked to each other by a sequence of0-100 nucleotides. Preferably, the sequence 5′-GGAG-3′ is linked to the3′ end of the sequence 5′-N1N2N3N4N5N6N7N8N9-3′.

In some embodiments, the sequence 5′-N1N2N3N4N5N6N7N8N9-3′ and thesequence 5′-GGAG-3′ are linked to each other by a sequence of 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 141, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, or 25 nucleotides.

In some embodiments, the oligonucleotide comprises a hairpin structurecomprising a hairpin loop of at least three nucleotides and wherein N4,N5, and N6 are in the loop region of the hairpin.

In some embodiments, the hairpin structure comprises a fully-doublestranded stem of at least four nucleotide basepairs.

In some embodiments, the sequence 5′-GGAG-3′ which is linked to the3′end of the stem of hairpin structure and there are be 0, 1, or2nucleotides between the 3′ end of the stem and 5′ end of the sequence5′ -GGAG-3′.

As used herein, the term “oligonucleotide” refers to a polymer oroligomer of nucleotide or nucleoside monomers consisting of naturallyoccurring bases, sugars and intersugar linkages. The term“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly.

The loop region of the hairpin structure can be at least three, e.g.four, five, six, seven, eight, nine, ten, eleven, or more nucleotides inlength. In some embodiments, the loop is from three to nine nucleotidesin length. In one embodiment, the loop is at least nine nucleotides inlength. In some embodiments, the loop comprises at least oneoligonucleotide modification described herein. Without limitations, anoligonucleotide modification can be present at an internal position ofthe loop or at one of the terminus positions of the loop.

Generally, the stem of the hairpin structure is fully double strandedand is at least four, e.g., four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen or more nucleotide basepairsin length. By “fully double-stranded” is meant that the double strandedregion does not comprise one or more single-stranded nucleotides in oneor both stands of the stem over its length. In other words, the stemdoes not comprise any interior loops, branch junctions or bulges. Insome embodiments, the stem comprises at least one oligonucleotidemodification described herein. Without limitations, an oligonucleotidemodification can be present at an internal position of the stem or atone of the terminus positions of the stem.

A part of the sequence of formula 5′-N1N2N3N4N5N6N7N8N9-3′ is present inthe loop of the hairpin structure. Preferably N4, N5, and N6 are presentin the loop. In some embodiments, N5 is at the third, fourth, fifth,sixth, or seventh of the loop. In one embodiment, N5 is at the middleposition of the loop, i.e., there are an equal number of single-strandednucleotides on both side of N5 in the loop. It is to be understood thatthe first position in the loop is the first nucleotide in the loop thatis not base-paired, counting from the end of stem. In some embodiments,the sequence of formula 5′-N1N2N3N4N5N6N7N8N9-3′ comprises at least oneoligonucleotide modification described herein. Without limitations, anoligonucleotide modification can be present at an internal position ofthe stem or at one of the terminus positions of the sequence of formula5′-N1N2N3N4N5N6N7N8N9-3′.

In some embodiments, N1 and N3 are independently selected purines andN7, N8, and N9 are independently selected pyrimidines.

In some embodiments, N2 and N4 are guanosine and N5 is adenosine.

In some embodiments, N1 and N5 are adenosine; N3 is adenosine oruridine; N2 and N4 are guanosine; and N6, N7, N8, and N9 are uridine.

In some embodiments, N1, N3, N8, and N9 are independently selectedpyrimidines and N7 is a purine.

In some embodiments, N1, N3, and N6 are uridine; N2, N5, and N7 areadenosine; N4 is guanosine; and N8 and N9 are cytosine.

Oligonucleotides of the present invention can be of various lengths. Insome embodiments, the oligonucleotide is at least 18 nucleotides inlength. In some embodiments, oligonucleotides can range from 19 to 100nucleotides in length. In some embodiments, the oligonucleotide is from19 to 50; 19 to 35; 19 to 30, or 19 to 25 nucleotides in length. In someembodiments, the oligonucleotide is 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides inlength.

In some embodiments, the oligonucleotide comprises, consists of orconsists essentially of the sequence 5′-GGGCAGAGAUUUUGCCCGGAG-3′ (SEQ IDNO: 16), 5′-GGGGUAGUGAUUUUACCCUGGAG-3′ (SEQ ID NO: 17) or5′-GGGGUCUAUGAUACCACCCCGGAG-3′ (SEQ ID NO: 18).

In some embodiments, the oligonucleotide is not one ofUUAGGGCAGGGAUUUUGCCCACAAGGAGGU (SEQ ID NO: 19), UAGAAUUACAUCAAGGGAGAU(SEQ ID NO: 20), GUGGGGUAGUGAUUUUACCCUGUUCAGGAGAU (SEQ ID NO: 21),UAGAGUUACACCCUGGGAGUU (SEQ ID NO: 22), UGGGGCUCUGCCCUGCUAUGGGAU (SEQ IDNO: 23), GGUCGGGUUGUGACAUUGCCCGCUGUGGAGAU (SEQ ID NO: 24),UUAGGGUCAUACCCCAUCUUGGAGAU (SEQ ID NO: 25),UGAGGGUCUAUGAUACCACCCGGUACAGGAGAU (SEQ ID NO: 26),UUAGGGUCACACCCACCACUGGGAGAU (SEQ ID NO: 27), GAGGAGGACACCCAAGGAGAUC (SEQID NO: 28), UCAGGGCAGUGAUGUUGCCCCUCGGAAGAU (SEQ ID NO: 29),GUGGGGUAGGGAUAUUAGGCCCCAAUUAGAAGAU (SEQ ID NO: 30),UAAGGGUCUGUGACACCACCCUCUGUUGGAGAU (SEQ ID NO: 31), orGGUAGGGUCURUGAYAYYRCCCGSURYRGGAGAU (SEQ ID NO: 32).

Unmodified oligonucleotides can be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. However, chemical modificationsto one or more of the subunits of oligonucleotide can confer improvedproperties, e.g., can render oligonucleotides more stable to nucleases.Typical oligonucleotide modifications can include one or more of: (i)alteration, e.g., replacement, of one or both of the non-linkingphosphate oxygens and/or of one or more of the linking phosphate oxygensin the phosphodiester intersugar linkage; (ii) alteration, e.g.,replacement, of a constituent of the ribose sugar, e.g., of the 2′hydroxyl on the ribose sugar; (iii) wholesale replacement of thephosphate moiety with “dephospho” linkers; (iv) modification orreplacement of a naturally occurring base with a non-natural base; (v)replacement or modification of the ribose-phosphate backbone, e.g.peptide nucleic acid (PNA); (vi) modification of the 3′ end or 5′ end ofthe oligonucleotide, e.g., removal, modification or replacement of aterminal phosphate group or conjugation of a moiety, e.g., conjugationof a ligand, to either the 3′ or 5′ end of oligonucleotide; and (vii)modification of the sugar, e.g., six membered rings.

The terms replacement, modification, alteration, and the like, as usedin this context, do not imply any process limitation, e.g., modificationdoes not mean that one must start with a reference or naturallyoccurring ribonucleic acid and modify it to produce a modifiedribonucleic acid bur rather modified simply indicates a difference froma naturally occurring molecule. As described below, modifications, e.g.,those described herein, can be provided as asymmetrical modifications.

An oligonucleotide described herein can comprise any oligonucleotidemodification described herein. In some embodiments, the oligonucleotidecomprises at least one modification. In some embodiments, themodification is selected from the group consisting of a sugarmodification, a non-phosphodiester intersugar (or internucleoside)linkage, nucleobase modification, and ligand conjugation. In someembodiments, the oligonucleotide comprises at least two differentmodifications selected from the group consisting of a sugarmodification, a non-phosphodiester intersugar linkage, nucleobasemodification, and ligand conjugation.

A modification described herein can be the sole modification, or thesole type of modification included on multiple nucleotides, or amodification can be combined with one or more other modificationsdescribed herein. The modifications described herein can also becombined onto an oligonucleotide, e.g. different nucleotides of anoligonucleotide have different modifications described herein.

The phosphate group in the intersugar linkage can be modified byreplacing one of the oxygens with a different substituent. One result ofthis modification to oligonucleotide phosphate intersugar linkages canbe increased resistance of the oligonucleotide to nucleolytic breakdown.Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. In some embodiments, one of the non-bridging phosphateoxygen atoms in the intersugar linkage can be replaced by any of thefollowing: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkylgroup, an aryl group, etc. . . . ), H, NR2 (R is hydrogen, optionallysubstituted alkyl, aryl), or OR (R is optionally substituted alkyl oraryl). The phosphorous atom in an unmodified phosphate group is achiral.However, replacement of one of the non-bridging oxygens with one of theabove atoms or groups of atoms renders the phosphorous atom chiral; inother words a phosphorous atom in a phosphate group modified in this wayis a stereogenic center. The stereogenic phosphorous atom can possesseither the “R” configuration (herein Rp) or the “S” configuration(herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligonucleotides diastereomers. Thus, whilenot wishing to be bound by theory, modifications to both non-bridgingoxygens, which eliminate the chiral center, e.g. phosphorodithioateformation, can be desirable in that they cannot produce diastereomermixtures. Thus, the non-bridging oxygens can be independently any one ofO, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either one of the linking oxygens or at both linking oxygens. Whenthe bridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Modified phosphate linkages where at least one of the oxygen linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non-phosphodiesterintersugar linkage” or “non-phosphodiester linker.”

The phosphate group can be replaced by non-phosphorus containingconnectors, e.g. dephospho linkers. Dephospho linkers are also referredto as non-phosphodiester linkers herein. While not wishing to be boundby theory, it is believed that since the charged phosphodiester group isthe reaction center in nucleolytic degradation, its replacement withneutral structural mimics should impart enhanced nuclease stability.Again, while not wishing to be bound by theory, it can be desirable, insome embodiment, to introduce alterations in which the charged phosphategroup is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include, butare not limited to, amides (for example amide-3 (3′-CH2-C(═O)—N(H)-5′)and amide-4 (3′-CH2-N(H)—C(═O)-5′)), hydroxylamino, siloxane(dialkylsiloxxane), carboxamide, carbonate, carboxymethyl, carbamate,carboxylate ester, thioether, ethylene oxide linker, sulfide,sulfonate,sulfonamide, sulfonate ester, thioformacetal (3′-S—CH2-O-5′), formacetal(3′-β-CH2-O-5), oxime, methyleneimino, methykenecarbonylamino,methylenemethylimino (MMI, 3′-CH2-N(CH3)-O-5′), methylenehydrazo,methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3′-O-C5′),thioethers (C3′-S-C5′), thioacetamido(C3-N(H)—C(═O)—CH2-S-C5′C3′-O—P(O)—O—SS—C5′, C3′-CH2-NH—NH—C5,3′-NHP(O)(OCH3)-O-5′ and 3′-NHP(O)(OCH3)-O-5′ and nonionic linkagescontaining mixed N, O, S and CH2 component parts. See for example,Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65).Preferred embodiments include methylenemethylimino (MMI),methylenecarbonylamino, amides, carbamate and ethylene oxide linker.

One skilled in the art is well aware that in certain instancesreplacement of a non-bridging oxygen can lead to enhanced cleavage ofthe intersugar linkage by the neighboring 2′-OH, thus in many instances,a modification of a non-bridging oxygen can necessitate modification of2′-OH, e.g., a modification that does not participate in cleavage of theneighboring intersugar linkage, e.g., arabinose sugar, 2′-O-alkyl, 2′-F,LNA and ENA.

Preferred non-phosphodiester intersugar linkages includephosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more enantiomeric excess of Spisomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer,phosphorodithioates, phsophotriesters, aminoalkylphosphotrioesters,alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates,phosphoramidates (e.g., N-alkylphosphoramidate), and boranophosphonates.

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiments, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themorpholino, cyclobutyl, pyrrolidine, peptide nucleic acid (PNA),aminoethylglycyl PNA (aegPNA) and backnone-extended pyrrolidine PNA(bepPNA) nucleoside surrogates. A preferred surrogate is a PNAsurrogate.

An oligonucleotide can include modification of all or some of the sugargroups of the nucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, n=1-50; “locked”nucleic acids (LNA) in which the oxygen at the 2′ position is connectedby (CH2)n, wherein n=1-4, to the 4′ carbon of the same ribose sugar,preferably n is 1 (LNA) or 2 (ENA); O-AMINE or O-(CH2)nAMINE (n=1-10,AMINE=NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, diheteroaryl amino, ethylene diamine orpolyamino); and O—CH2CH2(NCH2CH2NMe2)2.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the single-strand overhangs); halo (e.g.,fluoro); amino (e.g. NH2; alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or aminoacid); NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino); —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; thioalkyl; alkyl;cycloalkyl; aryl; alkenyl and alkynyl, which can be optionallysubstituted with e.g., an amino functionality.

A modification at the 2′ position can be present in the arabinoseconfiguration The term “arabinose configuration” refers to the placementof a substituent on the C2′ of ribose in the same configuration as the2′-OH is in the arabinose.

Oligonucleotides can also include abasic sugars, which lack a nucleobaseat C-1′ or have other chemical groups in place of a nucleobase at C1′.See for example U.S. Pat. No. 5,998,203, contents of which are hereinincorporated in their entirety. These abasic sugars can also be furthercontaining modifications at one or more of the constituent sugar atoms.Oligonucleotides can also contain one or more sugars that are the Lisomer, e.g. L-nucleosides. Modification to the sugar group can alsoinclude replacement of the 4′-O with a sulfur, optionally substitutednitrogen or CH2 group. In some embodiments, linkage between Cl' andnucleobase is in the α configuration. Sugar modifications can alsoinclude acyclic nucleotides, wherein a C—C bonds between ribose carbons(e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-04′, C1′-O4′) is absent and/or atleast one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′ or O4′)are independently or in combination absent from the nucleotide.

In some embodiments, the sugar modification is selected from the groupconsisting of 2′-H (DNA), 2′-O-Me (2′-O-methyl), 2′-O-MOE(2′-O-methoxyethyl), 2′-F, 2′-O-[2-(methylamino)-2-oxoethyl] (2′-O-NMA),2′-S-methyl, 2′-O—CH2-(4′-C) (LNA), 2′-O—CH2CH2-(4′-C) (ENA),2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE),2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′-F arabinose, 2′-OMe arabinose, arabinose, and anycombinations thereof.

It is to be understood that when a particular nucleotide is linkedthrough its 2′-position to the next nucleotide, the sugar modificationsdescribed herein can be placed at the 3′-position of the sugar for thatparticular nucleotide, e.g., the nucleotide that is linked through its2′ -position. A modification at the 3′ position can be present in thexylose configuration The term “xylose configuration” refers to theplacement of a substituent on the C3′ of ribose in the sameconfiguration as the 3′-OH is in the xylose sugar.

Adenine, cytosine, guanine, thymine and uracil are the most common bases(or nucleobases) found in nucleic acids. These bases can be modified orreplaced to provide oligonucleotides having improved properties. Forexample, nuclease resistant oligonucleotides can be prepared with thesebases or with synthetic and natural nucleobases (e.g., inosine,xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) andany one of the above modifications. Alternatively, substituted ormodified analogs of any of the above bases and “universal bases” can beemployed. When a natural base is replaced by a non-natural and/oruniversal base, the nucleotide is said to comprise a modified nucleobaseand/or a nucleobase modification herein. Modified nucleobase and/ornucleobase modifications also include natural, non-natural and universalbases, which comprise conjugated moieties, e.g. a ligand describedherein. Preferred conjugate moieties for conjugation with nucleobasesinclude cationic amino groups which can be conjugated to the nucleobasevia an appropriate alkyl, alkenyl or a linker with an amide linkage.

An oligonucleotide can also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as inosine, xanthine, hypoxanthine, nubularine,isoguanisine, tubercidine, 2-(halo)adenine, 2-(alkyl)adenine,2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine,6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine,8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine,8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine,8-(thioalkyl)adenine, 8-(thiol)adenine, N6-(isopentyl)adenine,N6-(methyl)adenine, N6,N6-(dimethyl)adenine, 2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine,7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine,8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine,8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine,8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine,3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine,5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine,5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(propynyl)cytosine,5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine,3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil,5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil,4-(thio)uracil, 5-(methyl)-4-(thio)uracil,5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil,5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil,5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil,5-(aminoallyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil,5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil,5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid,5-(methoxycarbonylmethyl)-2-(thio)uracil,5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil),2-(thio)pseudouracil,4-(thio)pseudouracil,2,4-(dithio)psuedouracil5-(alkyl)pseudouracil,5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil,5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil,5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil,1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil,1-substituted 2,4-(dithio)pseudouracil,1-(aminocarbonylethylenyl)-pseudouracil,1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phen thiazin-1-yl,1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine,nubularine, tubercidine, isoguanisine, inosinyl, 2-aza-inosinyl,7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl,nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl,3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl,3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,6-(methyl)-7-(aza)indolyl, imidizopyridinyl,9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, difluorotolyl,4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substitutedpyrimidines, N2-substituted purines, N6-substituted purines,O6-substituted purines, substituted 1,2,4-triazoles,pyrrolo-pyrimidin-2-on-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl,pyridopyrimidin-3-yl, 2-oxo-7-amino-pyridopyrimidin-3-yl,2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylatedderivatives thereof. Alternatively, substituted or modified analogs ofany of the above bases and “universal bases” can be employed.

As used herein, a universal nucleobase is any modified or nucleobasethat can base pair with all of the four naturally occurring nucleobaseswithout substantially affecting the melting behavior, recognition byintracellular enzymes or activity of the oligonucleotide duplex. Someexemplary universal nucleobases include, but are not limited to,2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine,4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3-methylisocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl,tetracenyl, pentacenyl, and structural derivatives thereof (see forexample, Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).

Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808;those disclosed in PCT App. Pub. No. WO/2009/120878; those disclosed inthe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990; those disclosedby English et al., Angewandte Chemie, International Edition, 1991, 30,613; those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijin, P.Ed. Wiley-VCH, 2008; and thosedisclosed by Sanghvi, Y. S., Chapter 15, dsRNA Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B., Eds., CRCPress, 1993. Contents of all of the above are herein incorporated byreference.

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.For example, the 3′ and/or 5′ ends of an oligonucleotide can beconjugated to other functional molecular entities such as labelingmoieties, e.g., fluorophores or protecting groups (based e.g., onsulfur, silicon, boron or ester). The functional molecular entities canbe attached to the sugar through a phosphate group and/or a linker. Theterminal atom of the linker can connect to or replace the linking atomof the phosphate group or the C-3′ or C-5′ O, N, S or C group of thesugar. Alternatively, the linker can connect to or replace the terminalatom of a nucleotide surrogate (e.g., PNAs).

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. Forexample, in some embodiments, the oligonucleotide is phosphorylated orincludes a phosphoryl analog at the 5′ terminus. Exemplary5′-modifications include, but are not limited to, 5′-monophosphate((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); 5′-alpha-thiotriphosphate;5′-beta-thiotriphosphate; 5′-gamma-thiotriphosphate; 5′-phosphoramidates((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′). Other 5′-modification include5′-alkylphosphonates (R(OH)(O)P—O-5′, R=alkyl, e.g., methyl, ethyl,isopropyl, propyl, etc. . . . ), 5′-alkyletherphosphonates(R(OH)(O)P—O-5′, R=alkylether, e.g., methoxymethyl (CH2OMe),ethoxymethyl, etc. . . . ). Other exemplary 5′-modifications includewhere Z is optionally substituted alkyl at least once, e.g.,((HO)2(X)P—O[—(CH2)a-O—P(X)(OH)—O]b-5′,((HO)2(X)P-O[—(CH2)a-P(X)(OH)-P]b-5′,((HO)2(X)P—[—(CH2)a-O—P(X)(OH)—O]b-5; dialkyl terminal phosphates andphosphate mimics: HO[—(CH2)a-O—P(X)(OH)—O]b-5′,H2[—(CH2)a-O—P(X)(OH)—O]b-5′, H[—(CH2)a-O—P(X)(OH)—O]b-5′,Me2NB[—(CH2)a-O—P(X)(OH)—O]b-5′, HO[—(CH2)a-P(X)(OH)—O]b-5′,H2N[—(CH2)a-P(X)(OH)—O]b-5′, H[—(CH2)a-P(X)(OH)—O]b-5′,Me2N[—(CH2)a-P(X)(OH)—O]b-5′, wherein a and b are each independently1-10. Other embodiments, include replacement of oxygen and/or sulfurwith BH3, BH3- and/or Se.

Additional terminal modifications are described, for example, inManoharan, M. et al. Antisense and Nucleic Acid Drug Development 12,103-128 (2002) and references therein.

In some embodiments, the oligonucleotide comprises a cap structure at 3′(3′-cap), 5′ (5′-cap) or both ends. In some embodiments, oligonucleotidecomprises a 3′-cap. In another embodiment, oligonucleotide comprises a5′-cap. In yet another embodiment, oligonucleotide comprises both a 3′cap and a 5′ cap. It is to be understood that when an oligonucleotidecomprises both a 3′ cap and a 5′ cap, such caps can be same or they canbe different.

As used herein, “cap structure” refers to chemical modifications, whichhave been incorporated at either terminus of oligonucleotide. See forexample U.S. Pat. No. 5,998,203 and International Patent PublicationWO03/70918, contents of which are herein incorporated in theirentireties. Exemplary 5′-caps include, but are not limited to, ligands,5′-5′-inverted nucleotide, 5′-5′-inverted abasic nucleotide residue,2′-5′ linkage, 5′-amino, 5′-amino-alkyl phosphate, 5′-hexylphosphate,5′-aminohexyl phosphate, bridging and/or non-bridging5′-phosphoramidate, bridging and/or non-bridging 5′-phosphorothioateand/or 5′-phosphorodithioate, bridging or non-bridging5′-methylphosphonate, non-phosphodiester intersugar linkage between theend two nucleotides, 4′,5′-methylene nucleotide,I-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotides, modified nucleobase nucleotide, phosphorodithioatelinkage, threo-pentofuranosyl nucleotide, acyclic nucleotide, acyclic3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide,5′-mercapto nucleotide and 5′-1,4-butanediol phosphate.

Exemplary 3′-caps include, but are not limited to, ligands,3′-3′-inverted nucleotide, 3′-3′-inverted abasic nucleotide residue,3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety,2′-5′-linkage, 3′-amino, 3′-amino-alkyl phosphate, 3′-hexylphosphate,3′-aminohexyl phosphate, bridging and/or non-bridging3′-phosphoramidate, bridging and/or non-bridging 3′-phosphorothioateand/or 3′-phosphorodithioate, bridging or non-bridging3′-methylphosphonate, non-phosphodiester intersugar linkage between theend two nucleotides, I-(beta-D-erythrofuranosyl) nucleotide, 4′-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,L-nucleotides, alpha-nucleotides, modified nucleobase nucleotide,phosphorodithioate linkage, threo-pentofuranosyl nucleotide, acyclicnucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic3,5-dihydroxypentyl nucleotide, and 3′-1,4-butanediol phosphate. Formore details see Beaucage and Iyer, 1993, Tetrahedron 49, 1925,incorporated by reference herein.

Other 3′ and/or 5′ caps amenable to the invention are described in Int.Pat. Pub. No. WO 2011/005861, content of which is incorporated herein byreference.

In some embodiments, the oligonucleotide comprises at least one (e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more), of 5′-5′, 3′-3′, 3′-2′,2′-5′, 2′-3′ or 2′-2′ intersugar linkage. In some embodiments, the lastnucleotide on the terminal end is linked via a 5′-5′, 3′-3′, 3′-2′,2′-5′, 2′-3′ or 2′-2′ intersugar linkage to the rest of theoligonucleotide. In some embodiments, the last nucleotide on both theterminal ends is linked via a 5′-5′, 3′-3′, 3′-2′, 2′-3′ or 2′-2′intersugar linkage to the rest of the oligonucleotide. In someembodiments, at least one 5′-5′, 3′-3′, 3′-2′, 2′-5′, 2′-3′ or 2′-2′intersugar linkage is a non-phosphodiester linkage.

A wide variety of entities, e.g., ligands, can be coupled to theoligonucleotides described herein. Ligands can include naturallyoccurring molecules, or recombinant or synthetic molecules. Exemplaryligands include, but are not limited to, polylysine (PLL), polyL-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydridecopolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleicanhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K,PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA),polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamidepolymers, polyphosphazine, polyethylenimine, cationic groups, spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin,melanotropin, lectin, glycoprotein, surfactant protein A, mucin,glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate,polyaspartate, aptamer, asialofetuin, hyaluronan, procollagen,immunoglobulins (e.g., antibodies), insulin, transferrin, albumin,sugar-albumin conjugates, intercalating agents (e.g., acridines),cross-linkers (e.g. psoralen, mitomycin C), porphyrins (e.g., TPPC4,texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA),lipophilic molecules (e.g, steroids, bile acids, cholesterol, cholicacid, adamantane acetic acid, 1-pyrene butyric acid,dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexylgroup, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecylgroup, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), peptides(e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cellpermeation peptide, endosomolytic/fusogenic peptide), alkylating agents,phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl,radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., naproxen, aspirin, vitamin E,folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies,hormones and hormone receptors, lectins, carbohydrates, multivalentcarbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitaminB, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamincofactors, lipopolysaccharide, an activator of p38 MAP kinase, anactivator of NF-κB, taxon, vincristine, vinblastine, cytochalasin,nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A,indanocine, myoservin, tumor necrosis factor alpha (TNFalpha),interleukin-1 beta, gamma interferon, natural or recombinant low densitylipoprotein (LDL), natural or recombinant high-density lipoprotein(HDL), and a cell-permeation agent (e.g., alpha-helical cell-permeationagent).

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides. Apeptidomimetic (also referred to herein as an oligopeptidomimetic) is amolecule capable of folding into a defined three-dimensional structuresimilar to a natural peptide. The peptide or peptidomimetic ligand canbe about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35,40, 45, or 50 amino acids long.

Exemplary amphipathic peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, cpf, bombinin-like peptide(blp), cathelicidins, ceratotoxins, s. clava peptides, hagfishintestinal antimicrobial peptides (hfiaps), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, h2a peptides, xenopus peptides,esculentinis-1, and caerins.

As used herein, the term “endosomolytic ligand” refers to moleculeshaving endosomolytic properties. Endosomolytic ligands promote the lysisof and/or transport of the composition of the invention, or itscomponents, from the cellular compartments such as the endosome,lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule,peroxisome, or other vesicular bodies within the cell, to the cytoplasmof the cell. Some exemplary endosomolytic ligands include, but are notlimited to, imidazoles, poly or oligoimidazoles, linear or branchedpolyethyleneimines (PEIs), linear and brached polyamines, e.g. spermine,cationic linear and branched polyamines, polycarboxylates, polycations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketals, orthoesters, linear or branched polymers with maskedor unmasked cationic or anionic charges, dendrimers with masked orunmasked cationic or anionic charges, polyanionic peptides, polyanionicpeptidomimetics, pH-sensitive peptides, natural and synthetic fusogeniclipids, natural and synthetic cationic lipids.

Exemplary endosomolytic/fusogenic peptides include, but are not limitedto, AALEALAEALEALAEALEALAEAAAAGGC (GALA) (SEQ ID NO: 33);AALAEALAEALAEALAEALAEALAAAAGGC (EALA) (SEQ ID NO: 34); ALEALAEALEALAEA(SEQ ID NO: 35); GLFEAIEGFIENGWEGMIWDYG (INF-7) (SEQ ID NO: 36);GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2) (SEQ ID NO: 37);GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7) (SEQ ID NO:38); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3) (SEQ ID NO:39); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF) (SEQ ID NO: 40);GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3) (SEQ ID NO: 41);GLFEAIEGFIENGWEGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n isnorleucine) (SEQ ID NO: 42); LFEALLELLESLWELLLEA (JTS-1) (SEQ ID NO:43); GLFKALLKLLKSLWKLLLKA (ppTG1) (SEQ ID NO: 44); GLFRALLRLLRSLWRLLLRA(ppTG20) (SEQ ID NO: 45); WEAKLAKALAKALAKHLAKALAKALKACEA (KALA) (SEQ IDNO: 46); GLFFEAIAEFIEGGWEGLIEGC (HA) (SEQ ID NO: 47);GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin) (SEQ ID NO: 48); HSWYG; andCHK6HC.

Without wishing to be bound by theory, fusogenic lipids fuse with andconsequently destabilize a membrane. Fusogenic lipids usually have smallhead groups and unsaturated acyl chains. Exemplary fusogenic lipidsinclude, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine(DOPE), phosphatidylethanolamine (POPE), andpalmitoyloleoylphosphatidylcholine (POPC).

Synthetic polymers with endosomolytic activity amenable to the presentinvention are described in U.S. Pat. App. Pub. No. 2009/0048410; No.2009/0023890; No. 2008/0287630; No. 2008/0287628; No. 2008/0281044; No.2008/0281041; No. 2008/0269450; No. 2007/0105804; No. 20070036865; andNo. 2004/0198687, content of all of which is incorporated herein byreference.

Exemplary cell permeation peptides include, but are not limited to,RQIKIWFQNRRMKWKK (penetratin) (SEQ ID NO: 49); GRKKRRQRRRPPQC (Tatfragment 48-60) (SEQ ID NO: 50); GALFLGWLGAAGSTMGAWSQPKKKRKV (signalsequence based peptide) (SEQ ID NO: 51); LLIILRRRIRKQAHAHSK (PVEC) (SEQID NO: 52); GWTLNSAGYLLKINLKALAALAKKIL (transportan) (SEQ ID NO: 53);KLALKLALKALKAALKLA (amphiphilic model peptide) (SEQ ID NO: 54);RRRRRRRRR (Arg9) (SEQ ID NO: 55); KFFKFFKFFK (Bacterial cell wallpermeating peptide) (SEQ ID NO: 56);LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37) (SEQ ID NO: 57);SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin P1) (SEQ ID NO: 58);ACYCRIPACIAGERRYGTCIYQGRLWAFCC (α-defensin) (SEQ ID NO: 59)DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (β-defensin) (SEQ ID NO: 60);RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39) (SEQ ID NO: 61);ILPWKWPWWPWRR-NH2 (indolicidin) (SEQ ID NO: 62); AAVALLPAVLLALLAP (RFGF)(SEQ ID NO: 63); AALLPVLLAAP (RFGF analogue) (SEQ ID NO: 64); andRKCRIVVIRVCR (bactenecin) (SEQ ID NO: 65).

Exemplary cationic groups include, but are not limited to, protonatedamino groups, derived from e.g., O-AMINE (AMINE=NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g.,O(CH2)nAMINE, (e.g., AMINE=NH2; alkylamino, dialkylamino, heterocyclyl,arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,ethylene diamine, polyamino); amino (e.g. NH2; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); and NH(CH2CH2NH)nCH2CH2-AMINE (AMINE=NH2;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino).

As used herein the term “targeting ligand” refers to any molecule thatprovides an enhanced affinity for a selected target, e.g., a cell, celltype, tissue, organ, region of the body, or a compartment, e.g., acellular, tissue or organ compartment. Some exemplary targeting ligandsinclude, but are not limited to, antibodies, antigens, folates, receptorligands, carbohydrates, aptamers, integrin receptor ligands, chemokinereceptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA,endothelin, GCPII, somatostatin, LDL and HDL ligands.

Carbohydrate based targeting ligands include, but are not limited to,D-galactose, multivalent galactose, N-acetyl-D-galactose (GalNAc),multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalentmannose, multivalent lactose, N-acetyl-galactosamine,N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacidsand lectins. The term multivalent indicates that more than onemonosaccharide unit is present. Such monosaccharide subunits can belinked to each other through glycosidic linkages or linked to a scaffoldmolecule.

A number of folate and folate analogs amenable to the present inventionas ligands are described in U.S. Pat. Nos. 2,816,110; 51410,104;5,552,545; 6,335,434 and 7,128,893, contents of which are hereinincorporated in their entireties by reference.

As used herein, the terms “PK modulating ligand” and “PK modulator”refers to molecules which can modulate the pharmacokinetics of thecomposition of the invention. Some exemplary PK modulator include, butare not limited to, lipophilic molecules, bile acids, sterols,phospholipid analogues, peptides, protein binding agents, vitamins,fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen,ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, andtransthyretia-binding ligands (e.g., tetraiidothyroacetic acid,2,4,6-triiodophenol and flufenamic acid). Oligonucleotides that comprisea number of phosphorothioate intersugar linkages are also known to bindto serum protein, thus short oligonucleotides, e.g. oligonucleotides ofcomprising from about 5 to 30 nucleiotides (e.g., 5 to 25 nulceotides,preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 nucleotides), and that comprise a plurality ofphosphorothioate linkages in the backbone are also amenable to thepresent invention as ligands (e.g. as PK modulating ligands). The PKmodulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more phosphorothioate and/orphosphorodithioate linkages. In some embodiments, all internucleotidelinkages in PK modulating oligonucleotide are phosphorothioate and/orphosphorodithioates linkages. In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also amenable to the presentinvention as PK modulating ligands. Binding to serum components (e.g.serum proteins) can be predicted from albumin binding assays, such asthose described in Oravcova, et al., Journal of Chromatography B (1996),677: 1-27.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

In some embodiments, the ligand is a fluorescent reporter, e.g. afluorophore. A wide variety of fluorescent reporter dyes are known inthe art. Typically, the fluorophore is an aromatic or heteroaromaticcompound and can be a pyrene, anthracene, naphthalene, acridine,stilbene, indole, benzindole, oxazole, thiazole, benzothiazole, cyanine,carbocyanine, salicylate, anthranilate, coumarin, fluorescein, rhodamineor other like compound. Suitable fluorescent reporters include xanthenedyes, such as fluorescein or rhodamine dyes, including, but not limitedto, Alexa Fluor® dyes (Invitrogen Corp.; Carlsbad, Calif.), fluorescein,fluorescein isothiocyanate (FITC), Oregon Green™, rhodamine, Texas red,tetrarhodamine isothiocynate (TRITC), 5-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G),N,N,N,N′-tetramefhyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine(ROX). Suitable fluorescent reporters also include the naphthylaminedyes that have an amino group in the alpha or beta position. Forexample, naphthylamino compounds include1-dimethylamino-naphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate,2-p-toluidinyl-6-naphthalene sulfonate, and5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS). Otherfluorescent reporter dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p(2-benzoxazolyl)phenyl)maleimide; cyanines, such as Cy2,indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-(-carboxy-pentyl)-3′ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H, 15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-inner salt (TR or TexasRed); BODIPY™ dyes; benzoxadiazoles; stilbenes; pyrenes; and the like.Many suitable forms of these fluorescent compounds are available and canbe used.

Ligands can be coupled to the oligonucleotides at various places, forexample, 3′-end, 5′-end, and/or at an internal position. When two ormore ligands are present, the ligand can be on opposite ends of anoligonucleotide. In preferred embodiments, the ligand is attached to theoligonucleotides via an intervening tether/linker. The ligand ortethered ligand can be present on a monomer when said monomer isincorporated into the growing strand. In some embodiments, the ligandcan be incorporated via coupling to a “precursor” monomer after said“precursor” monomer has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether (i.e.,having no associated ligand), e.g., monomer-linker-NH2 can beincorporated into a growing oligonucleotide strand. In a subsequentoperation, i.e., after incorporation of the precursor monomer into thestrand, a ligand having an electrophilic group, e.g., apentafluorophenyl ester or aldehyde group, can subsequently be attachedto the precursor monomer by coupling the electrophilic group of theligand with the terminal nucleophilic group of the precursor monomer'stether.

In another example, a monomer having a chemical group suitable fortaking part in Click Chemistry reaction can be incorporated e.g., anazide or alkyne terminated tether/linker. In a subsequent operation,i.e., after incorporation of the precursor monomer into the strand, aligand having complementary chemical group, e.g. an alkyne or azide canbe attached to the precursor monomer by coupling the alkyne and theazide together.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. When a ligand is conjugated to anucleobase, the preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing.

Conjugation to sugar moieties of nucleosides can occur at any carbonatom. Example carbon atoms of a sugar moiety that can be attached to aconjugate moiety include the 2′, 3′, and 5′ carbon atoms. The 1′position can also be attached to a conjugate moiety, such as in anabasic residue. Internucleosidic linkages can also bear conjugatemoieties. For phosphorus-containing linkages (e.g., phosphodiester,phosphorothioate, phosphorodithiotate, phosphoroamidate, and the like),the conjugate moiety can be attached directly to the phosphorus atom orto an O, N, or S atom bound to the phosphorus atom. For amine- oramide-containing internucleosidic linkages (e.g., PNA), the conjugatemoiety can be attached to the nitrogen atom of the amine or amide or toan adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative U.S. patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,149,782; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662; 5,688,941;5,714,166; 6,153,737; 6,172,208; 6,300,319; 6,335,434; 6,335,437;6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631; 6,559,279;contents which are herein incorporated in their entireties by reference.

In some embodiments, the ligands, e.g. endosomolytic ligands, targetingligands or other ligands, are linked to a monomer which is thenincorporated into the growing oligonucleotide strand during chemicalsynthesis. Such monomers are also referred to as carrier monomersherein. The carrier monomer is a cyclic group or acyclic group;preferably, the cyclic group is selected from the group consisting ofpyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, [1,3]-dioxolane, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclicgroup is selected from serinol backbone or diethanolamine backbone. Insome embodiments, the cyclic carrier monomer is based on pyrrolidinylsuch as 4-hydroxyproline or a derivative thereof

Exemplary ligands and ligand conjugated monomers amenable to theinvention are described in U.S. Pat. App. Pub. No. 2005/0107325;No.2005/0164235; No. 2005/0256069; No. 2006/0008822; No. 2005/0288244;No. 2007/0054279; Ser. No. 11/944,227; No. 2009/0239814; and No.2009/0247614, content of all of which is incorporated herein byreference. Ligands and ligand conjugated monomers amenable to theinvention are also described in

PCT App. Pub. No. WO/2004/065601; No WO/2004/090108; No. WO2004/091515;No. WO/2006/078278; No. WO/2006/073458; No. WO/2006/112872; No.WO/2008/131419; No. WO/2009/018332; No. WO/2009/073809; and No.WO/2009/126933, content of which is incorporated herein by reference.

In some embodiments, the covalent linkages between the oligonucleotideand other components, e.g. a ligand or a ligand carrying monomer can bemediated by a linker. This linker can be cleavable linker ornon-cleavable linker, depending on the application. As used herein, a“cleavable linker” refers to linkers that are capable of cleavage undervarious conditions. Conditions suitable for cleavage can include, butare not limited to, pH, UV irradiation, enzymatic activity, temperature,hydrolysis, elimination and substitution reactions, redox reactions, andthermodynamic properties of the linkage. In some embodiments, acleavable linker can be used to release the oligonucleotide aftertransport to the desired target. The intended nature of the conjugationor coupling interaction, or the desired biological effect, willdetermine the choice of linker group.

As used herein, the term “linker” means an organic moiety that connectstwo parts of a compound. Linkers typically comprise a direct bond or anatom such as oxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO,SO₂, SO₂NH or a chain of atoms, such as substituted or unsubstitutedalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, where one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R¹)₂, C(O), cleavable linking group,substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted heterocyclic; where IV ishydrogen, acyl, aliphatic or substituted aliphatic.

In some embodiments, the linker comprises at least one cleavable linkinggroup.

In some embodiments, the linker is a branched linker. The branchpoint ofthe branched linker may be at least trivalent, but can be a tetravalent,pentavalent or hexavalent atom, or a group presenting such multiplevalencies. In some embodiments, the branchpoint is , —N, —N(Q)-C, —O—C,—S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or —N(Q)C(O)O—C;wherein Q is independently for each occurrence H or optionallysubstituted alkyl. In some embodiments, the branchpoint is glycerol orderivative thereof.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood or serum of a subject, orunder a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; amidases; endosomes or agents that cancreate an acidic environment, e.g., those that result in a pH of five orlower; enzymes that can hydrolyze or degrade an acid cleavable linkinggroup by acting as a general acid, peptidases (which can be substratespecific) and proteases, and phosphatases.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In some embodiments, cleavable linking group is cleaved at least 1.25,1.5, 1.75, 2, 3, 4, 5, 10, 25, 50, or 100 times faster in the cell (orunder in vitro conditions selected to mimic intracellular conditions) ascompared to blood or serum (or under in vitro conditions selected tomimic extracellular conditions). In some embodiments, the cleavablelinking group is cleaved by less than 90%, 80%, 70%, 60%, 50%, 40%, 30%,20%, 10%, 5%, or 1% in the blood (or in vitro conditions selected tomimic extracellular conditions) as compared to in the cell (or under invitro conditions selected to mimic intracellular conditions)

Exemplary cleavable linking groups include, but are not limited to,redox cleavable linking groups (e.g., —S—S— and —C(R)₂—S—S—, wherein Ris H or C₁-C₆ alkyl and at least one R is C₁-C₆ alkyl such as CH₃ orCH₂CH₃); phosphate-based cleavable linking groups (e.g., —O—P(O)(OR)—O—,—O—P(S)(OR)—O—, —O—P(S)(SR)—O—, —S—P(O)(OR)—O—, —O—P(O)(OR)—S—,—S—P(O)(OR)—S—, —O—P(S)(ORk)-S—, —S—P(S)(OR)—O—, —O—P(O)(R)—O—,—O—P(S)(R)—O—, —S—P(O)(R)—O—, —S—P(S)(R)—O—, —S—P(O)(R)—S—,—O—P(S)(R)—S—, —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—,—S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—,—S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—,—S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—, wherein R is optionallysubstituted linear or branched C₁-C₁₀ alkyl); acid celavable linkinggroups (e.g., hydrazones, esters, and esters of amino acids, —C═NN— and—OC(O)—); ester-based cleavable linking groups (e.g., —C(O)O—);peptide-based cleavable linking groups, (e.g., linking groups that arecleaved by enzymes such as peptidases and proteases in cells, e.g.,—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids). A peptide based cleavable linking groupcomprises two or more amino acids. In some embodiments, thepeptide-based cleavage linkage comprises the amino acid sequence that isthe substrate for a peptidase or a protease found in cells.

In some embodiments, an acid cleavable linking group is cleavable in anacidic environment with a pH of about 6.5 or lower (e.g., about 6.0,5.5, 5.0, or lower), or by agents such as enzymes that can act as ageneral acid.

In some embodiments, pairings which increase the propensity to form aduplex are used at one or more of the positions in the double-stranded.The terminus base pair of the stem and the subsequent two base pairingpositions in the duplex are preferred for placement of modifications toincrease the propensity to form a duplex. It is preferred that at leastone, and more preferably two or three of the pairs of the recitedregions be chosen independently from the group of: G:C, a pair having ananalog that increases stability over Watson-Crick matches (A:T, A:U,G:C), 2-amino-A:U, 2-thio-U or 5-Me-thio-U:A, G-clamp (an analog of Chaving 4 hydrogen bonds):G, guanadinium-G-clamp:G, pseudo uridine:A, abase pair in which one or both subunits have a sugar modification, e.g.,a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding. Insome embodiments, at least one, at least, at least 2, or at least 3, ofthe base pairs promote duplex stability.

In some embodiments, at least one end of the stem double-stranded regionis terminated by a G:C, G:U, G-clamp:G or guanadinium-G-clamp:G basepair, i.e., the terminus base pair is a G:C, G:U, G-clamp:G orguanadinium-G-clamp:G base pair. In some embodiments, the G:C, G:U,G-clamp:G or guanadinium-G-clamp:G base pair encloses the loop of thehairpin structure of the oligonucleotide, i.e., the loop is terminatedby a G:C, G:U, G-clamp:G or guanadinium-G-clamp:G base pair. In someembodiments, the end of the stem away from the loop is terminated by aG:C, G:U, G-clamp:G or guanadinium-G-clamp:G base pair. In someembodiments, both ends of the stem are terminated independently by aG:C, G:U, G-clamp:G or guanadinium-G-clamp:G base pair.

G-clamps and guanidinium G-clamps are discussed in the followingreferences: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-ClampContaining Oligonucleotides and Their Inhibition of the HIV-1 Tat-TARInteraction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262,2003; Holmes et al., “Steric inhibition of human immunodeficiency virustype-1 Tat-dependent trans-activation in vitro and in cells byoligonucleotides containing 2′-O-methyl G-clamp ribonucleosideanalogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al.,“Structural basis for recognition of guanosine by a synthetic tricycliccytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta,86:966-978, 2003; Rajeev, et al., “High-Affinity Peptide Nucleic AcidOligomers Containing Tricyclic Cytosine Analogues,” Organic Letters,4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- andGuanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002;Maier et al., “Nuclease resistance of oligonucleotides containing thetricyclic cytosine analogues phenoxazine and9-(2-aminoethoxy)-phenoxazine (“G-clamp”) and origins of their nucleaseresistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al.,“A cytosine analog that confers enhanced potency to antisenseoligonucleotides,” PNAS, US, 96:3513-8, 1999.

The oligonucleotides described herein can be synthesized with solidphase synthesis, see for example “Oligonucleotide synthesis, a practicalapproach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides andAnalogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991(especially Chapter 1, Modern machine-aided methods ofoligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotidesynthesis, Chapter 3, 2′-O-Methyloligoribonucleotides: synthesis andapplications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5,Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesisof oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

The preparation of phosphinate oligonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligonucleotides is described in U.S.Pat. Nos. 5,256,775 or 5,366,878. The preparation of phosphotriesteroligonucleotides is described in U.S. Pat. No. 5,023,243. Thepreparation of boranophosphate oligonucleotide is described in U.S. Pat.Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-aminophosphoramidate oligonucleotides is described in U.S. Pat. No.5,476,925. 3′-Deoxy-3′-methylenephosphonate oligonucleotides isdescribed in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.Preparation of sulfur bridged nucleotides is described in Sproat et al.Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. TetrahedronLett. 1989, 30, 4693.

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32,301-310).

Methylenemethylimino linked oligonucleosides, also identified herein asMMI linked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified herein as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identifiedherein as amide-3 linked oligonucleosides, and methyleneaminocarbonyllinked oligonucleosides, also identified herein as amide-4 linkedoligonucleosides as well as mixed intersugar linkage compounds having,as for instance, alternating MMI and PO or PS linkages can be preparedas is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inInternational Application Nos. PCT/US92/04294 and PCT/US92/04305(published as WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligonucleosides can be prepared as is describedin U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linkedoligonucleosides can be prepared as is described in U.S. Pat. No.5,223,618. Siloxane replacements are described in Cormier, J. F. et al.Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are describedin Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 1 1972, 1991. Carbamate replacements are described in Stirchak,E. P. Nucleic Acids Res. 1989, 17, 6129.

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They can also be prepared in accordance with U.S. Pat.No. 5,539,083.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,457,191; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200;6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062;6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is hereinincorporated by reference in its entirety.

As oligonucleotides are polymers of subunits or monomers, many of themodifications described herein can occur at a position which is repeatedwithin an oligonucleotide, e.g., a modification of a nucleobase, asugar, a phosphate moiety, or the non-bridging oxygen of a phosphatemoiety. It is not necessary for all positions in a given oligonucleotideto be uniformly modified, and in fact more than one of theaforementioned modifications can be incorporated in a singleoligonucleotide or even at a single nucleoside within anoligonucleotide.

In some cases the modification will occur at all of the subjectpositions in the oligonucleotide but in many, and in fact in most casesit will not. By way of example, a modification can occur at a 3′ or 5′terminal position, can occur in the internal region, can occur in 3′, 5′or both terminal regions, e.g. at a position on a terminal nucleotide orin the last 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from an end of theoligonucleotide. In some embodiments, the terminal nucleotide (e.g.,3′-terminal or preferably 5′-terminal) does not comprise a modification.

Provided herein also are methods for regulating miRNA biogenesis. In oneaspect, the invention provides a method for promoting miRNA processingof pre-miRNA to a mature miRNA in a cell, the method comprisingcontacting a cell with an oligonucleotide described herein.

The term “contacting” or “contact” as used herein in connection withcontacting a cell includes subjecting the cell to an appropriate culturemedia which comprises the indicated Ephexin5 inhibitor. Where the cellis in vivo, “contacting” or “contact” includes administering theoligonucleotide in a pharmaceutical composition to a subject via anappropriate administration route such that oligonucleotide contacts thecell in vivo.

For in vivo methods, a therapeutically effective amount of anoligonucleotide can be administered to a subject. Methods ofadministering compounds to a subject are known in the art and easilyavailable to one of skill in the art.

Provided herein also are methods for the treatment and/or prevention ofcancer by administering to a subject an oligonucleotide describedherein.

Subjects amenable to treatment using the methods as disclosed hereininclude subjects at risk of a cancer, as well as subjects at risk ofdeveloping cancer. In some embodiments, subjects amenable to treatmentusing the methods as disclosed herein include subjects identified withor having increased risk of cancer, for example subjects identified tocarry a genetic mutation or polymorphism associated with an increasedrisk of developing cancer. Such mutations and genetic susceptibilitygenes and loci are commonly known by persons skilled in the art, forexample some of the more commonly known genes where a mutation isassociated with increase in cancer include, but are not limited to;BRAC1, BRAC2, EGFR, EIF4A2, ERBB2, RBI, CDKN2A, P53, INK4a, APC, MLH1,MSH2, MSH6, WTI, NF1, NF2, and VHL (see world-wide web at web site:cancer.org/docroot/ETO/content/ETO 14x_oncogenes_and_tumor_suppressor_genes-dot-asp).

In some embodiments, subjects can be screened for their likelihood ofhaving or developing cancer based on a number of biochemical and geneticmarkers or other biomarkers. Biomarkers are defined as cellular,biochemical, molecular or genetic alterations by which a normal,abnormal or simply biologic process can be recognized or monitored.Biomarkers are measurable in biological media, such as human tissues,cells or fluids. Biomarkers could be used to identify pathologicalprocesses before individuals become symptomatic or to identifyindividuals who are susceptible to cancer.

Several classes of biomarkers in cancer cells and bodily fluids havebeen studied, mostly in laboratories examining specific observations butalso in limited clinical settings. Several biomarkers have shown onlylimited utility: e.g., CD44, telomerase, transforming growth factor-α(TGFα), transforming growth factor-β (TGF-β), epidermal growth factorreceptor erbB-2 (erbB-2), epidermal growth factor receptor erbB-3(erbB-3), mucin 1 (MUC1), mucin 2 (MUC2) and cytokeratin 20 (CK20).Other biomarkers are used in clinical practice and include, for exampleProstate specific antigen (PSA) and cancer antibody or tumor marker 125(CA125). Several protein markers can be used as cancer biomarkers, forexample but not limited to, Fecal occult blood test (FOBT), which is aprotein biomarker shown to decrease cause-specific mortality in cancerscreens.

In one embodiment, subjects amenable to treatment using the methods asdisclosed herein include subjects with a high level of Lin28 in abiological sample from the subject as compared to a reference level ofLin-28, and thus have reduced processing of tumor suppressor miRNAs,such as let-7 miRNA. In some embodiments, the subject is assessed ifthey are at risk of having cancer by identifying the level of Lin28 in abiological sample from the subject and comparing the level of Lin28 witha reference level of Lin-28. For example, if the level of Lin28 in abiological sample from the subject is above a reference level, thesubject is at risk of having a metastasis or a malignant cancer. In someembodiments, the biological sample obtained from the subject is from abiopsy tissue sample, and in some embodiments, the sample is from atumor or cancer tissue sample. The level of Lin28 can be determined byany method known by one of ordinary skill in the art, for example bynorthern blot analysis or RT-PCR for mRNA expression levels, or ELISA orwestern blot analysis for protein expression levels.

In some embodiments, a reference level of Lin28 is the level of Lin28that does not result in malignancy or a malignant cancer. In someembodiments, the reference level of Lin28 the based on the level ofLin28 expression or protein activity in a normal tissue sample, where inthe tissue sample is a biological tissue sample from a tissue matched,species matched and age matched biological sample. In some embodiments,the reference level of Lin28 is based on a biological sample is from anon-malignant matched tissue sample. In some embodiments, the referencelevel of Lin28 is based on a biological sample from normal tissue, forexample non-cancer tissue, or a non-stem cell cancer tissue sample.

In alternative embodiments, a subject amenable to treatment using themethods as disclosed herein include subjects with a low level of Let-7miRNA family members in a biological sample from the subject as comparedto a reference level of Let-7 miRNA, and thus have reduced suppressionof oncogenes expression. In some embodiments, the subject is assessed ifthey are at risk of having cancer by identifying the level of let-7miRNA in a biological sample from the subject and comparing the level oflet-7 miRNA with a reference level of let-7 miRNA. For example, if thelevel of let-7 miRNA in a biological sample from the subject is below areference level, the subject is at risk of having a metastasis or amalignant cancer. In some embodiments, the biological sample obtainedfrom the subject is from a biopsy tissue sample, and in someembodiments, the sample is from a tumor or cancer tissue sample. Thelevel of let-7 miRNA can be determined by methods known by the skilledartisan, for example by northern blot analysis or RT-PCR. In someembodiments, the reference level of let-7 miRNA is the level of let-7miRNA that does not result in malignancy or a malignant cancer. In someembodiments, the reference level of let-7 miRNA is the based on thelevel of let-7 miRNA expression in a normal tissue sample, where in thetissue sample is a biological tissue sample from a tissue matched,species matched and age matched biological sample. In some embodiments,the reference level of let-7 miRNA is based on a biological sample isfrom a non-malignant matched tissue sample. In some embodiments, thereference level let-7 miRNA is based on a biological sample from anon-stem cell cancer tissue sample.

In some embodiments, the biological sample obtained from the subject isfrom a biopsy tissue sample. In some embodiments, the biological sampleis from a tumor or cancer tissue sample.

A subject administered an oligonucleotide inhibiting Lin28 activity asdisclosed herein can be evaluated for symptoms relative to a subject notadministered the oligonucleotide. A measurable change in the severity asymptom (i.e., a decrease in at least one symptom, i.e. 10% or greaterdecrease), or a delay in the onset of a symptom, in animals treated withan oligonucleotide described herein versus untreated animals isindicative of therapeutic efficacy.

In some embodiments, the method as disclosed herein are useful for thetreatment of any disease or disorder characterized by lack or reducedexpression of tumor suppressor miRNAs, for example but not limited tolet-7 family miRNAs.

In alternative embodiments, the methods as disclosed herein are usefulfor the treatment of any disease or disorder characterized by increasedof Lin28 as compared to a reference level.

In alternative embodiments, the methods as disclosed herein are usefulfor the treatment of any disease or disorder characterized by a decreasein let-7 miRNA as compared to a reference level.

In some embodiments, the subject is assessed if they have decreasedlevel of let-7 miRNA in a biological sample from the subject as comparedto a reference level of let-7 miRNA in a reference biological sample.

In some embodiments, compositions and methods described herein can beused for the treatment of adult and/or pediatric oncology including insolid phase tumors/malignancies, locally advanced tumors, human softtissue sarcomas, metastatic cancer, including lymphatic metastases,blood cell malignancies including multiple myeloma, acute and chronicleukemias, and lymphomas, head and neck cancers including mouth cancer,larynx cancer and thyroid cancer, lung cancers including small cellcarcinoma and non-small cell cancers, breast cancers including smallcell carcinoma and ductal carcinoma, gastrointestinal cancers includingesophageal cancer, stomach cancer, colon cancer, colorectal cancer andpolyps associated with colorectal neoplasia, pancreatic cancers, livercancer, urologic cancers including bladder cancer and prostate cancer,malignancies of the female genital tract including ovarian carcinoma,uterine (including endometrial) cancers, and solid tumor in the ovarianfollicle, kidney cancers including renal cell carcinoma, brain cancersincluding intrinsic brain tumors, neuroblastoma, askocytic brain tumors,gliomas, metastatic tumor cell invasion in the central nervous system,bone cancers including osteomas, skin cancers including malignantmelanoma, tumor progression of human skin keratinocytes, squamous cellcarcinoma, basal cell carcinoma, hemangiopericytoma and Kaposi's sarcoma

Cancers include, but are not limited to, bladder cancer; breast cancer;brain cancer including glioblastomas and medulloblastomas; cervicalcancer; choriocarcinoma; colon cancer including colorectal carcinomas;endometrial cancer; esophageal cancer; gastric cancer; head and neckcancer; hematological neoplasms including acute lymphocytic andmyelogenous leukemia, multiple myeloma, AIDS associated leukemias andadult T-cell leukemia lymphoma; intraepithelial neoplasms includingBowen's disease and Paget's disease, liver cancer; lung cancer includingsmall cell lung cancer and non-small cell lung cancer; lymphomasincluding Hodgkin's disease and lymphocytic lymphomas; neuroblastomas;oral cancer including squamous cell carcinoma; osteosarcomas; ovariancancer including those arising from epithelial cells, stromal cells,germ cells and mesenchymal cells; pancreatic cancer; prostate cancer;rectal cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,liposarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skincancer including melanomas, Kaposi's sarcoma, basocellular cancer, andsquamous cell cancer; testicular cancer including germinal tumors suchas seminoma, non-seminoma (teratomas, choriocarcinomas), stromal tumors,and germ cell tumors; thyroid cancer including thyroid adenocarcinomaand medullar carcinoma; transitional cancer and renal cancer includingadenocarcinoma and Wilm's tumor.

In one embodiment, compositions and methods described herein can be usedfor treatment or prevention of breast cancer. In some embodiments,compositions and methods described herein can be used for treatment orprevention of, for example but not limited to; lung cancer, hepaticcancer or leukemia, for example but not limited to lung carcinoma,chronic myelogenous leukemia (CML) and HCC (hepatic cell carcinoma).

In addition, compositions and methods as disclosed herein can also beused for prophylactic treatment of cancer. There are hereditaryconditions and/or environmental situations (e.g. exposure tocarcinogens) known in the art that predispose an individual todeveloping cancers, or subjects identified to have increased expressionof Lin28 as compared to a reference sample. Under these circumstances,it may be beneficial to treat these individuals with therapeuticallyeffective doses of an agent which inhibit Lin28 and/or Lin-28B to reducethe risk of developing cancers.

In one embodiment, the compositions and methods disclosed herein areuseful for a subject who has cancer regression.

In another embodiment, the compositions and methods disclosed herein areuseful for a subject who has a therapy resistant cancer, for example achemotherapy resistant cancer.

In some embodiments, the compositions and methods disclosed herein areuseful for a subject who has cancer and has been exposed to adjuvantcancer therapies.

In another embodiment, the compositions and methods disclosed herein areuseful for a subject with a malignant cancer. In some embodiments, thecompositions and methods disclosed herein are useful for a subject witha cancer or tumor comprising a cancer stem cell.

Most therapeutic strategies for cancer are aimed at reducing oreliminating the tumor or tumor. In some embodiments, the compositionsand methods disclosed herein are also useful in the treatment of otherdisease or disorders associated with abnormal cellular proliferation ordifferentiation of stem cells. Thus, treatment can be directed to asubject who is affected but asymptomatic with cancer, for example, adisease of an organ or tissue in a subject characterized by poorlycontrolled or uncontrolled multiplication of normal or abnormal cells inthat tissue and its effect on the body as a whole.

Cancer diseases which can be treated or prevented by the compositionsand methods disclosed herein include, but are not limited to, benignneoplasms, dysplasias, hyperplasias as well as neoplasms showingmetastatic growth or any other transformations like e.g. leukoplakiaswhich often precede a breakout of cancer.

Cancer therapy can also include prophylaxis, including agents which slowor reduce the risk of cancer in a subject. In other embodiments, acancer therapy is any treatment or any means to prevent theproliferation of cells with abnormal proliferation or cancerous cells.In some embodiments, then anti-cancer treatment is an agent whichsuppresses the EGF-EGFR pathway, for example but not limited toinhibitors and agents of EGFR. Inhibitors of EGFR include, but are notlimited to, tyrosine kinase inhibitors such as quinazolines, such as PID153035, 4-(3-chloroanilino) quinazoline, orCP-358,774,pyridopyrimidines, pyrimidopyrimidines, pyrrolopyrimidines, such as CGP59326, CGP 60261 and CGP 62706, and pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines (Traxleretal., (1996) J.Med Chem 39:2285-2292), curcumin (diferuloyl methane) (Laxmin arayana,et al., (1995), Carcinogen 16:1741-1745), 4,5-bis(4-fluoroanilino)phthalimide (Buchdunger et al. (1995) Clin. Cancer Res.1:813-821; Dinney et al. (1997) Clin. Cancer Res. 3:161-168);tyrphostins containing nitrothiophene moieties (Brunton et al. (1996)Anti Cancer Drug Design 11:265-295); the protein kinase inhibitor ZD-1839 (AstraZeneca); CP-358774 (Pfizer, Inc.); PD-01 83805(Warner-Lambert), EKB-569 (Torrance et al., Nature Medicine, Vol. 6, No.9, September 2000, p. 1024), HKI-272 and HKI-357 (Wyeth); or asdescribed in International patent application WO05/018677 (Wyeth);W099/09016 (American Cyanamid); W098/43960 (American Cyanamid); WO98/14451; WO 98/02434; W097/38983 (Warener Labert); W099/06378 (WarnerLambert); W099/06396 (Warner Lambert) ; W096/30347 (Pfizer, Inc.);W096/33978 (Zeneca); W096/33977 (Zeneca); and W096/33980 (Zeneca), WO95/19970; U.S. Pat. App. Nos. 2005/0101618 assigned to Pfizer,2005/0101617, 20050090500 assigned to OSI Pharmaceuticals, Inc.; allherein incorporated by reference. Further useful EGFR inhibitors aredescribed in U.S. Pat. App. No. 20040127470, particularly in tables 10,11, and 12, and are herein incorporated by reference.

In another embodiment, the present invention encompasses combinationtherapy in which subjects identified as having, or increased risk ofdeveloping cancer are administered an anti-cancer combination therapywhere combinations of anti-cancer agents are used are used incombination with cytostatic agents, anti-VEGF and/or p53 reactivationagent. A cytostatic agent is any agent capable of inhibiting orsuppressing cellular growth and multiplication. Examples of cytostaticagents used in the treatment of cancer are paclitaxel, 5-fluorouracil,5-fluorouridine, mitomycin-C, doxorubicin, and zotarolimus. Other cancertherapeutics include inhibitors of matrix metalloproteinases such asmarimastat, growth factor antagonists, signal transduction inhibitorsand protein kinase C inhibitors.

In another embodiment, the anti-cancer therapy includes achemotherapeutic regimen further comprises radiation therapy. In analternate embodiment, the therapy comprises administration of ananti-EGFR antibody or biological equivalent thereof.

In some embodiments, the anti-cancer treatment comprises theadministration of a chemotherapeutic drug selected from the groupconsisting of fluoropyrimidine (e.g., 5-FU), oxaliplatin, CPT-11, (e.g.,irinotecan) a platinum drug or an anti EGFR antibody, such as thecetuximab antibody or a combination of such therapies, alone or incombination with surgical resection of the tumor. In yet a furtheraspect, the treatment compresses radiation therapy and/or surgicalresection of the tumor masses. In one embodiment, the present inventionencompasses administering to a subject identified as having, orincreased risk of developing RCC an anti-cancer combination therapywhere combinations of anti-cancer agents are used, such as for exampleTaxol, cyclophosphamide, cisplatin, gancyclovir and the like.Anti-cancer therapies are well known in the art and are encompassed foruse in the methods of the present invention. Chemotherapy includes, butis not limited to an alkylating agent, mitotic inhibitor, antibiotic, orantimetabolite, anti-angiogenic agents etc. The chemotherapy cancomprise administration of CPT-11, temozolomide, or a platin compound.Radiotherapy can include, for example, x-ray irradiation, w-irradiation,y-irradiation, or microwaves.

The term “chemotherapeutic agent” or “chemotherapy agent” are usedinterchangeably herein and refers to an agent that can be used in thetreatment of cancers and neoplasms, for example brain cancers andgliomas and that is capable of treating such a disorder. In someembodiments, a chemotherapeutic agent can be in the form of a prodrugwhich can be activated to a cytotoxic form. Chemotherapeutic agents arecommonly known by persons of ordinary skill in the art and areencompassed for use in the present invention. For example,chemotherapeutic drugs for the treatment of tumors and gliomas include,but are not limited to: temozolomide (Temodar), procarbazine (Matulane),and lomustine (CCNU). Chemotherapy given intravenously (by IV, vianeedle inserted into a vein) includes vincristine (Oncovin or VincasarPFS), cisplatin (Platinol), carmustine (BCNU, BiCNU), and carboplatin(Paraplatin), Mexotrexate (Rheumatrex orTrexall), irinotecan (CPT-11);erlotinib; oxalipatin; anthracyclins-idarubicin and daunorubicin;doxorubicin; alkylating agents such as melphalan and chlorambucil;cisplatinum, methotrexate, and alkaloids such as vindesine andvinblastine.

Some examples of anti-VEGF agents include bevacizumab (AvastinTM), VEGFTrap, CP-547,632, AG13736, AG28262, SU5416, SU11248, SU6668, ZD-6474,ZD4190, CEP-7055, PKC 412, AEE788, AZD-2171, sorafenib, vatalanib,pegaptanib octasodium, IM862, DC101, angiozyme, Sirna-027, caplostatin,neovastat, ranibizumab, thalidomide, and AGA-1470, a synthetic analog offumagillin (alternate names: Amebacilin, Fugillin, Fumadil B, Fumadil)(A. G. Scientific, catalog #F1028), an angio-inhibitory compoundsecreted by Aspergillus fumigates.

As used herein the term “anti-VEGF agent” refers to any compound oragent that produces a direct effect on the signaling pathways thatpromote growth, proliferation and survival of a cell by inhibiting thefunction of the VEGF protein, including inhibiting the function of VEGFreceptor proteins. Exemplary VEGF inhibitors, i.e., anti-VEGF agents,include for example, AVASTIN® (bevacizumab), an anti-VEGF monoclonalantibody of Genentech, Inc. of South San Francisco, Calif., VEGF Trap(Regeneron/Aventis). Additional VEGF inhibitors include CP-547,632(3-(4Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride;Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, &SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca),CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788(Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; BayerPharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known asPTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptaniboctasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(glufanide disodium, Cytran Inc. of Kirkland, Wash., USA),VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.),angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) andChiron (Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,Sirna Therapeutics, San Francisco, Calif.) Caplostatin, solubleectodomains of the VEGF receptors, Neovastat (AEterna Zentaris Inc;Quebec City, Calif.) and combinations thereof.

For administering to a subject, the Lin28 fragment or an oligonucleotidedescribed herein can be formulated in a pharmaceutically acceptablecomposition. Thus, in another aspect, the present invention providespharmaceutically acceptable compositions which comprise atherapeutically-effective amount of a Lin28 fragment or anoligonucleotide described herein, formulated together with one or morepharmaceutically acceptable carriers (additives) and/or diluents. Thepharmaceutical composition can be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), gavages, lozenges, dragees,capsules, pills, tablets (e.g., those targeted for buccal, sublingual,and systemic absorption), boluses, powders, granules, pastes forapplication to the tongue; (2) parenteral administration, for example,by subcutaneous, intramuscular, intravenous or epidural injection as,for example, a sterile solution or suspension, or sustained-releaseformulation; (3) topical application, for example, as a cream, ointment,or a controlled-release patch or spray applied to the skin; (4)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (5) sublingually; (6) ocularly; (7) transdermally; (8)transmucosally; or (9) nasally. Additionally, the oligonucleotide can beimplanted into a patient or injected using a drug delivery system. See,for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236(1984); Lewis, ed. “Controlled Release of Pesticides andPharmaceuticals” (Plenum Press, New York, 1981); U.S. Pat. No.3,773,919; and U.S. Pat. No. 35 3,270,960, contents of all of which areincorporated herein by reference.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C2-C12 alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compound ofthe present invention which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment.Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other agents that inhibit pathologicalprocesses in neurodegenerative disorders.

A formulated oligonucleotide composition can assume a variety of states.In some examples, the composition is at least partially crystalline,uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20,or 10% water). In another example, the oligonucleotide is in an aqueousphase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., beincorporated into a delivery vehicle, e.g., a liposome (particularly forthe aqueous phase) or a particle (e.g., a micro particle as can beappropriate for a crystalline composition). Generally, theoligonucleotide composition is formulated in a manner that is compatiblewith the intended method of administration.

In particular embodiments, the composition is prepared by at least oneof the following methods: spray drying, lyophilization, vacuum drying,evaporation, fluid bed drying, or a combination of these techniques; orsonication with a lipid, freeze-drying, condensation and otherself-assembly.

An oligonucleotide preparation can be formulated in combination withanother agent, e.g., another therapeutic agent or an agent thatstabilizes the oligonucleotide, e.g., a protein that complex witholigonucleotide to form an oligonucleotide-protein complex. Still otheragents include chelators, e.g., EDTA (e.g., to remove divalent cationssuch as Mg²⁺), salts, DNAse inhibitors, RNAse inhibitors (e.g., a broadspecificity RNAse inhibitor such as RNAsin) and so forth.

The oligonucleotides can be formulated in liposomes. As used herein, aliposome is a structure having lipid-containing membranes enclosing anaqueous interior. Liposomes can have one or more lipid membranes. Insome embodiments, liposomes have an average diameter of less than about100 nm. More preferred embodiments provide liposomes having an averagediameter from about 30-70 nm and most preferably about 40-60 nm.Oligolamellar large vesicles and multilamellar vesicles have multiple,usually concentric, membrane layers and are typically larger than 100nm. Liposomes with several nonconcentric membranes, i.e., severalsmaller vesicles contained within a larger vesicle, are termedmultivesicular vesicles.

Liposomes can further comprise one or more additional lipids and/orother components such as sterols, e.g., cholesterol. Additional lipidscan be included in the liposome compositions for a variety of purposes,such as to prevent lipid oxidation, to stabilize the bilayer, to reduceaggregation during formation or to attach ligands onto the liposomesurface. Any of a number of additional lipids and/or other componentscan be present, including amphipathic, neutral, cationic, anioniclipids, and programmable fusion lipids. Such lipids and/or componentscan be used alone or in combination. One or more components of theliposome can comprise a ligand, e.g., a targeting ligand.

Liposome compositions can be prepared by a variety of methods that areknown in the art. See e.g., U.S. Pat. Nos. 4,235,871; 4,737,323;4,897,355 and 5,171,678; published International Applications WO96/14057 and WO 96/37194; Felgner, P. L. et al., Proc. Natl. Acad. Sci.,USA (1987) 8:7413-7417, Bangham, et al. M. Mol. Biol. (1965) 23:238,Olson, et al. Biochim. Biophys. Acta (1979) 557:9, Szoka, et al. Proc.Natl. Acad. Sci. (1978) 75: 4194, Mayhew, et al. Biochim. Biophys. Acta(1984) 775:169, Kim, et al. Biochim. Biophys. Acta (1983) 728:339, andFukunaga, et al. Endocrinol. (1984) 115:757.

The oligonucleotides of the invention can be prepared and formulated asmicelles. As used herein, “micelles” are a particular type of molecularassembly in which amphipathic molecules are arranged in a sphericalstructure such that all hydrophobic portions on the molecules aredirected inward, leaving the hydrophilic portions in contact with thesurrounding aqueous phase. The converse arrangement exists if theenvironment is hydrophobic.

In some embodiments, the formulations comprises micelles formed from anoligonucleotide of the invention and at least one amphiphilic carrier,in which the micelles have an average diameter of less than about 100nm, preferably. More preferred embodiments provide micelles having anaverage diameter less than about 50 nm, and even more preferredembodiments provide micelles having an average diameter less than about30 nm, or even less than about 20 nm.

Micelle formulations can be prepared by mixing an aqueous solution ofthe oligonucleotide composition, an alkali metal C₈ to C₂₂ alkylsulphate, and an amphiphilic carrier. The amphiphilic carrier can beadded at the same time or after addition of the alkali metal alkylsulphate. Micelles will form with substantially any kind of mixing ofthe ingredients but vigorous mixing in order to provide smaller sizemicelles.

The oligonucleotides of the present invention can be prepared andformulated as emulsions. As used herein, “emulsion” is a heterogeneoussystem of one liquid dispersed in another in the form of droplets.Emulsions are often biphasic systems comprising two immiscible liquidphases intimately mixed and dispersed with each other. Either of thephases of the emulsion can be a semisolid or a solid, as is the case ofemulsion-style ointment bases and creams. The oligonucleotide can bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase.

In some embodiments, the compositions are formulated as microemulsions.As used herein, “microemulsion” refers to a system of water, oil andamphiphile which is a single optically isotropic and thermodynamicallystable liquid solution. Microemuslions also include thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature, for example see Idson, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245; and Block, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 335, contents of which are hereinincorporated by reference in their entirety.

The oligonucleotides of the present invention can be prepared andformulated as lipid particles, e.g., formulated lipid particles (FLiPs)comprising (a) an oligonucleotide of the invention, where saidoligonucleotide has been conjugated to a lipophile and (b) at least onelipid component, for example an emulsion, liposome, isolatedlipoprotein, reconstituted lipoprotein or phospholipid, to which theconjugated oligonucleotide has been aggregated, admixed or associated.The stoichiometry of oligonucleotide to the lipid component can be 1:1.Alternatively the stoichiometry can be 1:many, many:1 or many:many,where many is two or more.

The FLiP can comprise triacylglycerols, phospholipids, glycerol and oneor several lipid-binding proteins aggregated, admixed or associated viaa lipophilic linker molecule with an oligonucleotide. Surprisingly, ithas been found that due to said one or several lipid-binding proteins incombination with the above mentioned lipids, the FLiPs show affinity toliver, gut, kidney, steroidogenic organs, heart, lung and/or muscletissue. These FLiPs can therefore serve as carrier for oligonucleotidesto these tissues. For example, lipid-conjugated oligonucleotides, e.g.,cholesterol-conjugated oligonucleotides, bind to HDL and LDL lipoproteinparticles which mediate cellular uptake upon binding to their respectivereceptors thus directing oligonucleotide delivery into liver, gut,kidney and steroidogenic organs, see Wolfrum et al. Nature Biotech.(2007), 25:1145-1157.

The FLiP can be a lipid particle comprising 15-25% triacylglycerol,about 0.5-2% phospholipids and 1-3% glycerol, and one or severallipid-binding proteins. FLiPs can be a lipid particle having about15-25% triacylglycerol, about 1-2% phospholipids, about 2-3% glycerol,and one or several lipid-binding proteins. In some embodiments, thelipid particle comprises about 20% triacylglycerol, about 1.2%phospholipids and about 2.25% glycerol, and one or several lipid-bindingproteins.

Another suitable lipid component for FLiPs is lipoproteins, for exampleisolated lipoproteins or more preferably reconstituted lipoproteins.Exemplary lipoproteins include chylomicrons, VLDL (Very Low DensityLipoproteins), IDL (Intermediate Density Lipoproteins), LDL (Low DensityLipoproteins) and HDL (High Density Lipoproteins). Methods of producingreconstituted lipoproteins are known in the art, for example see A.Jones, Experimental Lung Res. 6, 255-270 (1984), U.S. Pat. Nos.4,643,988 and 5,128,318, PCT publication WO87/02062, Canadian Pat. No.2,138,925. Other methods of producing reconstituted lipoproteins,especially for apolipoproteins A-I, A-II, A-IV, apoC and apoE have beendescribed in A. Jonas, Methods in Enzymology 128, 553-582 (1986) and G.Franceschini et al. J. Biol. Chem., 260(30), 16321-25 (1985).

One preferred lipid component for FLiP is Intralipid. Intralipid® is abrand name for the first safe fat emulsion for human use. Intralipid®20% (a 20% intravenous fat emulsion) is made up of 20% soybean oil, 1.2%egg yolk phospholipids, 2.25% glycerin, and water for injection. It isfurther within the present invention that other suitable oils, such assafflower oil, can serve to produce the lipid component of the FLiP.

FLiP can range in size from about 20-50 nm or about 30-50 nm, e.g.,about 35 nm or about 40 nm. In some embodiments, the FLiP has a particlesize of at least about 100 nm. FLiPs can alternatively be between about100-150 nm, e.g., about 110 nm, about 120 nm, about 130 nm, or about 140nm, whether characterized as liposome- or emulsion-based. Multiple FLiPscan also be aggregated and delivered together; therefore the size can belarger than 100 nm.

The process for making the lipid particles comprises the steps of: (a)mixing a lipid component with one or several lipophile (e.g.cholesterol) conjugated oligonucleotides that can be chemicallymodified; and (b) fractionating this mixture. In some embodiments, theprocess comprises the additional step of selecting the fraction withparticle size of 30-50 nm, preferably of about 40 nm in size.

Some exemplary lipid particle formulations amenable to the invention aredescribed in U.S. Pat. App. Pub. No. 2010/0003317, content of which isincorporated herein by reference.

In some embodiments, the oligonucleotide is formulated in yeast cellwall particles (“YCWP”). A yeast cell wall particle comprises anextracted yeast cell wall exterior and a core, the core comprising apayload (e.g., oligonucleotides). Exterior of the particle comprisesyeast glucans (e.g. beta glucans, beta-1,3-glucans, beta-1,6-glucans),yeast mannans, or combinations thereof. Yeast cell wall particles aretypically spherical particles about 1-4 μm in diameter.

Preparation of yeast cell wall particles is known in the art, and isdescribed, for example in U.S. Pat. Nos. 4,992,540; 5,082,936;5,028,703; 5,032,401; 5,322,841; 5,401,727; 5,504,079; 5,607,677;5,741,495; 5,830,463; 5,968,811; 6,444,448; and 6,476,003, U.S. Pat.App. Pub. Nos. 2003/0216346 and 2004/0014715, and Int. App. Pub. No. WO2002/12348, contents of which are herein incorporated by reference intheir entirety. Applications of yeast cell like particles for drugdelivery are described, for example in U.S. Pat. No. 5,032,401;5,607,677; 5,741,495; and 5,830,463, and U.S. Pat. Pub Nos. 2005/0281781and 2008/0044438, contents of which are herein incorporated by referencein their entirety. U.S. Pat. App. Pub. No. 2009/0226528, contents ofwhich are herein incorporated by reference, describes formulation ofnucleic acids with yeast cell wall particles for delivery ofoligonucleotide to cells.

Exemplary formulations for oligonucleotides are described in U.S. Pat.Nos. 4,897,355: 4,394,448; 4,235,871; 4,231,877; 4,224,179; 4753,788,4,673:567, 4,247,411: 4,814,270; 5,567,434; 5,552,157; 5,565,213;5,738,868; 5,795,587; 5,922,859: and 6,077,663, lnt. App Nos.PCT/US07/079203, filed Sep. 21, 2007; PCT/US07/080331, filed Oct. 3,2007; U.S. patent application Ser. No. 12/123,922, filed May 28, 2008;U.S. Pat. App. Pub. No. 2006/0240093 and No. 2007/0135372, contents ofwhich are herein incorporated by reference in their entirety. Bair(1994) Bioconjugate Chem 5:382-389, and Lewis et al. (1996) PNAS93:3176-3181), also describe formulations for oligonucleotides that areamenable to the invention, contents of which are herein incorporated byreference in their entirety.

The phrase “therapeutically-effective amount” as used herein means thatamount of an oligonucleotide described herein or a compositioncomprising an oligonucleotide described herein which is effective forproducing some desired therapeutic effect in at least a sub-populationof cells in an animal at a reasonable benefit/risk ratio applicable toany medical treatment. For example, an amount of an oligonucleotideadministered to a subject that is sufficient to produce a statisticallysignificant, measurable inhibition of a Lin28 polypeptide activity orproduce a statistically significant, measurable increase in processingof a pre-miRNA.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. Routes of administration suitable forthe methods of the invention include both local and systemicadministration. Generally, local administration results in more of thecomposition being delivered to a specific location as compared to theentire body of the subject, whereas, systemic administration results indelivery to essentially the entire body of the subject.

An oligonucleotide can be administered by any appropriate route known inthe art including, but not limited to, oral or parenteral routes,including intravenous, intramuscular, subcutaneous, transdermal, airway(aerosol), pulmonary, nasal, rectal, and topical (including buccal andsublingual) administration. Exemplary modes of administration include,but are not limited to, injection, infusion, instillation, inhalation,or ingestion. “Injection” includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some embodiments, the compositions areadministered by intravenous infusion or injection.

As used herein, a “subject” means a human or animal. Examples ofsubjects include primates (e.g., humans, and monkeys).Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female.

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofconditions or disorders associated with decreased spine/excitatorysynapse formation and/or numbers. In addition, the methods andcompositions described herein can be used to treat domesticated animalsand/or pets.

The amount of a Lin28 fragment or an oligonucleotide that can becombined with a carrier material to produce a single dosage form willgenerally be that amount of the compound that produces a therapeuticeffect. Generally out of one hundred percent, this amount will rangefrom about 0.01% to 99% of the compound, preferably from about 5% toabout 70%, most preferably from 10% to about 30%.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices, are preferred.

As used herein, the term ED denotes effective dose and is used inconnection with animal models. The term EC denotes effectiveconcentration and is used in connection with in vitro models.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage liespreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage may vary within this rangedepending upon the dosage form employed and the route of administrationutilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The dosage may be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that an oligonucleotide is given at a dose from 1μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1 μg/kgto 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to 100mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg,1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood thatranges given here include all intermediate ranges, for example, therange 1 mg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg,8 mg/kg to 10 mg/kg, 9 mg/kg to10 mg/kg , and the like. It is to be further understood that the rangesintermediate to the given above are also within the scope of thisinvention, for example, in the range 1 mg/kg to 10 mg/kg, dose rangessuch as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg , andthe like.

In some embodiments, the compositions are administered at a dosage sothat oligonucleotide has an in vivo concentration of less than 500 nM,less than 400 nM, less than 300 nM, less than 250 nM, less than 200 nM,less than 150 nM, less than 100 nM, less than 50 nM, less than 25 nM,less than 20, nM, less than 10 nM, less than 5 nM, less than 1 nM, lessthan 0.5 nM, less than 0.1 nM, less than 0.05, less than 0.01, nM, lessthan 0.005 nM, or less than 0.001 nM after 15 mins, 30 mins, 1 hr, 1.5hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10hrs, 11 hrs, 12 hrs or more of time of administration.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to oligonucleotide. The desired dose can beadministered every day or every third, fourth, fifth, or sixth day. Thedesired dose can be administered at one time or divided into subdoses,e.g., 2-4 subdoses and administered over a period of time, e.g., atappropriate intervals through the day or other appropriate schedule.Such sub-doses can be administered as unit dosage forms. In someembodiments of the aspects described herein, administration is chronic,e.g., one or more doses daily over a period of weeks or months. Examplesof dosing schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months or more.

The oligonucleotide can be administrated to a subject in combinationwith one or more pharmaceutically active agents. Exemplarypharmaceutically active compound include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., NY; Physicians' DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete content of all of which are herein incorporated inits entirety.

The oligonucleotide and the pharmaceutically active agent can beadministrated to the subject in the same pharmaceutical composition orin different pharmaceutical compositions (at the same time or atdifferent times). When administrated at different times, theoligonucleotide and the pharmaceutically active agent can beadministered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2hours, 3 hours, 4, hours, 8 hours, 12 hours, 24 hours of administrationof the other. When the oligonucleotide and the pharmaceutically activeagent are administered in different pharmaceutical compositions, routesof administration can be different.

In some embodiments, the pharmaceutically active agent is an anti-canceragent.

The invention can also be described by any one of the following numberedparagraphs:

-   -   1. An isolated RNA oligonucleotide comprising:        -   a. a nucleotide sequence of formula            5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′, wherein N², N⁴, and N⁵ are            independently a purine; N⁶ is a pyrimidine; N¹, N³, N⁷, N⁸,            and N⁹ are independently any nucleotide; and        -   b. a nucleotide sequence of 5′-GGAG-3′, wherein the sequence            5′-GGAG-3′ is linked to the 3′ of the sequence of formula            5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′, wherein the sequence 5′-GGAG-3′ is            single-stranded,        -   wherein there are from 0 to 100 nucleotides between the 3′            end of 5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′ and 5′ end of the sequence            5′-GGAG-3′.    -   2. The oligonucleotide of paragraph 1, wherein the        oligonucleotide comprises a hairpin structure comprising a        hairpin loop of at least 3 nucleotides and N⁴, N⁵, and N⁶ are in        the loop region of the hairpin.    -   3. The oligonucleotide of paragraph 2, wherein the hairpin        structure comprises a double-stranded stem of at least four        nucleotide base pairs, wherein the stem is fully        double-stranded.    -   4. The oligonucleotide of any of paragraphs 1-3, wherein the        oligonucleotide is from 19 to 100 nucleotides in length.    -   5. The oligonucleotide of any of paragraphs 3-4, wherein the        stem comprises at least one G-clamp:G or guanadinium-G-clamp:G        base pair.    -   6. The oligonucleotide of any of paragraphs 3-5, wherein the        stem is terminated by a G:C, G:U, G-clamp:G or        guanadinium-G-clamp:G base pair.    -   7. The oligonucleotide of any of paragraphs 1-6, wherein the        oligonucleotide comprises at least one 5′-5′, 3′-3′, 3′-2′,        2′-5′, 2′-3′ or 2′-2′ intersugar.    -   8. The oligonucleotide of any of paragraphs 1-6, wherein the        oligonucleotide comprises at least one modification selected        from the group consisting of a sugar modification, a        non-phosphodiester intersugar (or internucleoside) linkage,        nucleobase modification, and ligand conjugation.    -   9. The oligonucleotide of any of paragraphs 1-8, wherein the        oligonucleotide comprises a sugar modification selected from the        group consisting of 2′-H (DNA), 2′-O-Me (2′-O-methyl), 2′-O-MOE        (2′-O-methoxyethyl), 2′-F, 2′-O[2-(methylamino)-2-oxoethyl]        (2′-O-NMA), 2′-S-methyl, 2′-O—CH2-(4′-C) (LNA),        2′-O—CH₂CH₂-(4′-C) (ENA), 2′-O-aminopropyl (2′-O-AP),        2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl        (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-F        arabinose, 2′-OMe arabinose, arabinose, and any combinations        thereof.    -   10. The oligonucleotide of paragraph 8 or 9, wherein the sugar        medication is located within the loop, at the 5′ or 3′ end of        the loop; within the stem; within the sequence 5′-GGAG-3′; or at        N¹, N², N³, N⁴, N⁵, N⁶, N⁷, N⁸, or N⁹.    -   11. The oligonucleotide of any of paragraphs 1-10, wherein the        oligonucleotide comprises at least one non-phosphodiester        intersugar linkage selected from the group consisting of        phosphorothioate, phosphorodithioate, alkyl-phosphonate and        phosphoramidate linkage.    -   12. The oligonucleotide of any of paragraphs 8-11, wherein the        at least one modified intersugar linkage is located within the        loop, at the 5′ or 3′ end of the loop; within the stem; within        the sequence 5′-GGAG-3′; at the 5′ position of the 5′ most        guanosine of the sequence 5′-GGAG-3′; or at 5′ or 3′ position of        N¹, N², N³, N⁴, N⁵, N⁶, N⁷, N⁸, or N⁹.    -   13. The oligonucleotide of any of paragraphs 1-12, wherein the        oligonucleotide comprises a nucleobase modification selected        from the group consisting of inosine, thymine, xanthine,        hypoxanthine, nubularine, isoguanisine, tubercidine,        2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine,        2-(amino)adenine, 2-(aminoalkyll)adenine,        2-(aminopropyl)adenine, 2-(methylthio)-N⁶-(isopentenyl)adenine,        6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine,        8-(alkenyl)adenine, 8-(alkyl)adenine, 8-(alkynyl)adenine,        8-(amino)adenine, 8-(halo)adenine, 8-(hydroxyl)adenine,        8-(thioalkyl)adenine, 8-(thiol)adenine, N⁶-(isopentyl)adenine,        N⁶-(methyl)adenine, N⁶,N⁶-(dimethyl)adenine,        2-(alkyl)guanine,2-(propyl)guanine, 6-(alkyl)guanine,        6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine,        7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine,        8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine,        8-(hydroxyl)guanine, 8-(thioalkyl)guanine, 8-(thiol)guanine,        N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine,        3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine,        5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine,        5-(propynyl)cytosine, 5-(propynyl)cytosine,        5-(trifluoromethyl)cytosine, 6-(azo)cytosine,        N⁴-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil,        2-(thio)uracil5-(methyl)-2-(thio)uracil,        5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil,        5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil,        5-(methyl)-2,4-(dithio)uracil,        5-(methylaminomethyl)-2,4-(dithio)uracil,        5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil,        5-(allylamino)uracil, 5-(aminoallyl)uracil,        5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil,        5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,        5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil,        5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid,        5-(methoxycarbonylmethyl)-2-(thio)uracil,        5-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil,        5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil,        dihydrouracil, N³-(methyl)uracil, 5-uracil (i.e., pseudouracil),        2-(thio)pseudouracil,4-(thio)pseudouracil,2,4-(dithio)psuedouracil,5-(alkyl)pseudouracil,        5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil,        5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil,        5-(methyl)-4-(thio)pseudouracil,        5-(alkyl)-2,4-(dithio)pseudouracil,        5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil,        1-substituted 2(thio)-pseudouracil, 1-substituted        4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil,        1-(aminocarbonylethylenyl)-pseudouracil,        1-(aminocarbonylethylenyl)-2(thio)-pseudouracil,        1-(aminocarbonylethylenyl)-4-(thio)pseudouracil,        1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,        1-(aminoalkylaminocarbonylethylenyl)-pseudouracil,        1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil,        1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil,        1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,        1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,        1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,        1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,        1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted        1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted        1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted        1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted        1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,        7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,        7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,        7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,        7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,        7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,        7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,        7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,        7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,        1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine,        hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl,        2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl,        nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl,        aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl,        5-(methyl)isocarbostyrilyl,        3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,        6-(methyl)-7-(aza)indolyl, imidizopyridinyl,        9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,        7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,        2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl,        4,6-(dimethyl)indolyl, phenyl, napthalenyl, anthracenyl,        phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl,        difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole,        A-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone,        5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine,        2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines,        N²-substituted purines, N⁶-substituted purines, O⁶-substituted        purines, substituted 1,2,4-triazoles, or any O-alkylated or        N-alkylated derivatives thereof.    -   14. The oligonucleotide of any of paragraphs 8-13, wherein the        nucleobase modification is located within the loop, at the 5′ or        3′ end of the loop; within the stem; within the sequence        5′-GGAG-3′; or at N¹, N², N³, N⁴, N⁵, N⁶, N⁷, N⁸, or N⁹.    -   15. The oligonucleotide of any of paragraphs 1-14, wherein the        oligonucleotide is conjugated with a ligand selected from the        group consisting of polylysine (PLL), poly L-aspartic acid, poly        L-glutamic acid, styrene-maleic acid anhydride copolymer,        poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic        anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer        (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K,        PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl        alcohol (PVA), polyurethane, poly(2-ethylacryllic acid),        N-isopropylacrylamide polymers, polyphosphazine,        polyethylenimine, cationic groups, spermine, spermidine,        polyamine, pseudopeptide-polyamine, peptidomimetic polyamine,        dendrimer polyamine, arginine, amidine, protamine, cationic        lipid, cationic porphyrin, quaternary salt of a polyamine,        thyrotropin, melanotropin, lectin, glycoprotein, surfactant        protein A, mucin, glycosylated polyaminoacids, transferrin,        bisphosphonate, polyglutamate, polyaspartate, aptamer,        asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g.,        antibodies), insulin, transferrin, albumin, sugar-albumin        conjugates, intercalating agents (e.g., acridines),        cross-linkers (e.g. psoralen, mitomycin C), porphyrins (e.g.,        TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons        (e.g., phenazine, dihydrophenazine), artificial endonucleases        (e.g., EDTA), lipophilic molecules (e.g, steroids, bile acids,        cholesterol, cholic acid, adamantane acetic acid, 1-pyrene        butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol,        geranyloxyhexyl group, hexadecylglycerol, borneol, menthol,        1,3-propanediol, heptadecyl group, palmitic acid, myristic        acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,        dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha        helical peptide, amphipathic peptide, RGD peptide, cell        permeation peptide, endosomolytic/fusogenic peptide), alkylating        agents, phosphate, amino, mercapto, polyamino, alkyl,        substituted alkyl, radiolabeled markers, enzymes, haptens (e.g.        biotin), transport/absorption facilitators (e.g., naproxen,        aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g.,        imidazole, bisimidazole, histamine, imidazole clusters,        acridine-imidazole conjugates, Eu3+ complexes of        tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies,        hormones and hormone receptors, lectins, carbohydrates,        multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E,        vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin        and pyridoxal), vitamin cofactors, lipopolysaccharide, an        activator of p38 MAP kinase, an activator of NF-κB, taxon,        vincristine, vinblastine, cytochalasin, nocodazole,        japlakinolide, latrunculin A, phalloidin, swinholide A,        indanocine, myoservin, tumor necrosis factor alpha (TNFalpha),        interleukin-1 beta, gamma interferon, natural or recombinant low        density lipoprotein (LDL), natural or recombinant high-density        lipoprotein (HDL), a cell-permeation agent (e.g., a.helical        cell-permeation agent), and any combinations thereof    -   16. The oligonucleotide of any of paragraphs 8-15, wherein the        ligand is conjugated at the 5′ end or 3′ end of the        oligonucleotide, in the loop, within the stem, within the        sequence 5′-GGAG-3′; or within the sequence        5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′.    -   17. The oligonucleotide of any of paragraphs 1-16, wherein the        oligonucleotide comprises a fluorescent reporter (e.g., a        fluorophore).    -   18. The oligonucleotide of paragraph 17, wherein the fluorescent        reporter is selected from the group consisting of fluorescein        dyes, rhodamine dyes, naphthylamine dyes, coumarins, acridines,        N-(p(2-benzoxazolyl)phenyl)maleimide; cyanines, 1H,5H,11H,        15H-Xantheno[2,3,4-ij: 5,6,7-i′j′]diquinolizin-18-ium; 9-[2(or        4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4(or        2)-sulfophenyl]-2,3,6,7,12,13,16,17octahydro-inner salt (TR or        Texas Red); BODIPY™ dyes; benzoxadiazoles; stilbenes; pyrenes;        and the like.    -   19. The oligonucleotide of paragraph 17 or 18, wherein the        fluorescent reporter is conjugated at the 5′ end or 3′ end of        the oligonucleotide, within the loop, within the stem, within        the sequence 5′-GGAG-3′; or within the sequence        5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-3′.    -   20. The oligonucleotide of any of paragraphs 1-18, wherein the        oligonucleotide comprises a monophosphate, diphosphate,        triphosphate, monothiophosphate (phosphorothioate);        monodithiophosphate (phosphorodithioate), phosphorothiolate;        alpha-thiotriphosphate; beta-thiotriphosphate;        gamma-thiotriphosphate; P-N phosphoramidate (HO)(NH₂)(O)P—O-5′);        5′O—N phosphoramidate; ((HO)₂(O)P—NH-5′); alkylphosphonate        (R(OH)(O)P—O-5′, wherein R is alkyl); and alkyletherphosphonate        (R(OH)(O)P—O-5′, wherein R is alkylether) at the 5′ end.    -   21. The oligonucleotide of any of paragraphs 1-20, wherein the        5′ end of the oligonucleotide is covalently linked to the 3′ end        of the oligonucleotide.    -   22. The oligonucleotide of any of paragraphs 1-21, wherein N¹        and N³ are independently selected purines and N⁷, N⁸, and N⁹ are        independently selected pyrimidines.    -   23. The oligonucleotide of any of paragraphs 1-22, wherein N²        and N⁴ are guanosine and N⁵ is adenosine.    -   24. The oligonucleotide of any of paragraphs 1-23, wherein N¹        and N⁵ are adenosine; N³ is adenosine or uridine; N² and N⁴ are        guanosine; and N⁶, N⁷, N⁸, and N⁹ are uridine.    -   25. The oligonucleotide of paragraph 22, wherein the        oligonucleotide comprises the sequence        5′-GGGCAGAGAUUUUGCCCGGAG-3′ (SEQ ID NO: 16) or        5′-GGGGUAGUGAUUUUACCCUGGAG-3′ (SEQ ID NO: 17).    -   26. The oligonucleotide of any of paragraphs 1-21, wherein N¹,        N³, N⁸, and N⁹ are independently selected pyrimidines and N⁷ is        a purine.    -   27. The oligonucleotide any of paragraphs 1-21 or 26, wherein        N¹, N³, and N⁶ are uridine; N², N⁵, and N⁷ are adenosine; N⁴ is        guanosine; and N⁸ and N⁹ are cytosine.    -   28. The oligonucleotide of paragraph 27, wherein the        oligonucleotide comprises the sequence        5′-GGGGUCUAUGAUACCACCCCGGAG-3′ (SEQ ID NO: 18).    -   29. The oligonucleotide of any of paragraphs 1-28, wherein the        oligonucleotide inhibits the activity of a Lin28 polypeptide    -   30. A pharmaceutical composition comprising a oligonucleotide of        any of paragraphs 1-29 and a pharmaceutically acceptable        carrier.    -   31. A method for promoting miRNA processing of pri-miRNA to        mature miRNA in a cell, the method comprising contacting a cell        with an isolated oligonucleotide of any of paragraphs 1-29.    -   32. The method of paragraph 31, wherein the mature miRNA is a        tumor suppressor miRNA.    -   33. The method of any of paragraphs 31 or 32, wherein the mature        miRNA is a member of the let-7 miRNA family.    -   34. The method of any of paragraphs 31-33, wherein the cell        comprises a cancer cell.    -   35. The method of paragraph 34, wherein the cancer cell        comprises a cancer cell line.    -   36. The method of paragraph 34 or 35, wherein the cancer cell is        a pre-cancer cell, a malignant cancer cell, a therapy resistant        cancer cell or a cancer stem cell.    -   37. The method of any of paragraphs 34-36, wherein the cancer        cell is selected from the group consisting of: a breast cancer        cell, a lung cancer cell, lung adrenocarcinoma cell, a head and        neck cancer cell, a bladder cancer cell, a chronic myelogenous        leukemia (CML) cell, a stomach cancer cell, a nervous system        cancer cell, a bone cancer cell, a bone marrow cancer cell, a        brain cancer cell, a colon cancer cell, a colorectal cancer        cell, a esophageal cancer cell, a endometrial cancer cell, a        gastrointestinal cancer cell, a genital-urinary cancer cell, a        stomach cancer cell, a lymphomas cell, a melanoma cell, a glioma        cell, a bladder cancer cell, a pancreatic cancer cell, a gum        cancer cell, a kidney cancer cell, a retinal cancer cell, a        liver cancer cell, a nasopharynx cancer cell, an ovarian cancer        cell, an oral cancer cell, a bladder cancer cell, a        hematological neoplasm cell, a follicular lymphoma cell, a        cervical cancer cell, a multiple myeloma cell, a B-cell chronic        lymphcylic leukemia cell, a B-cell lymphoma cell, an        osteosarcoma cell, a thyroid cancer cell, a prostate cancer        cell, a colon cancer cell, a prostate cancer cell, a skin cancer        cell, a stomach cancer cell, a testis cancer cell, a tongue        cancer cell, an uterine cancer cell, and any combinations        thereof.    -   38. The method of any of paragraphs 31-37, wherein the cell is a        human cell.    -   39. The method of any of paragraphs 31-38, wherein said contact        is in vitro, in vivo, in a subject or ex vivo.    -   40. The method of any of paragraphs 39, wherein the in vivo        contact is in a subject, which subject is identified to have, or        be at risk of an increase in the level of expression and/or        activity of Lin-28 or the subject is identified to have, or be        at risk of a reduction of the level or expression and/or        activity, or loss of expression of a tumor suppressor miRNA.    -   41. The method of paragraph 39 or 40, wherein the in vivo        contact is in a human.    -   42. A method of treating or preventing a cancer in a subject,        comprising administering to a subject an effective amount of an        isolated oligonucleotide of any of paragraphs 1-29.    -   43. The method of paragraph 42, wherein the subject is        identified to have, or be at risk of an increase in the level of        expression and/or activity of Lin28 or the subject is identified        to have, or be at risk of a reduction of the level or expression        and/or activity, or loss of expression of a tumor suppressor        miRNA.    -   44. The method of paragraph 42 or 43, further comprising a        diagnosing a subject for ac cancer prior to administrating the        oligonucleotide.    -   45. The method of any of paragraphs 42-44, wherein the tumor        suppressor miRNA is a member of the let-7 miRNA family.    -   46. The method of any of the paragraphs 42-45, wherein the        cancer is a pre-cancer, malignant cancer, therapy resistant        cancer or a cancer comprising cancer stem cells.    -   47. The method of any of the paragraphs 42-46, wherein the        cancer is selected from the group consisting of breast cancer,        lung cancer, head and neck cancer, bladder cancer, stomach        cancer, cancer of the nervous system, bone cancer, bone marrow        cancer, brain cancer, colon cancer, colorectal cancer,        esophageal cancer, endometrial cancer, gastrointestinal cancer,        genital-urinary cancer, stomach cancer, lymphomas, melanoma,        glioma, bladder cancer, pancreatic cancer, gum cancer, kidney        cancer, retinal cancer, liver cancer, nasopharynx cancer,        ovarian cancer, oral cancers, bladder cancer, hematological        neoplasms, follicular lymphoma, cervical cancer, multiple        myeloma, B-cell chronic lymphcylic leukemia, B-cell lymphoma,        osteosarcomas, thyroid cancer, prostate cancer, colon cancer,        prostate cancer, skin cancer, stomach cancer, testis cancer,        tongue cancer, or uterine cancer.    -   48. The method of any of paragraphs 42-47, wherein said        administering is intravenous, intradermal, intramuscular,        intraarterial, intralesional, percutaneous, subcutaneous, or by        aerosol.    -   49. The method of any of paragraphs 42-48, further comprising        administering to the subject one or more additional therapies.    -   50. The method of paragraph 49, wherein additional therapies are        selected from the group consisting of surgery, chemotherapy,        radiotherapy, thermotherapy, immunotherapy, hormone therapy or        laser therapy.    -   51. The method of any of the paragraphs 42-50, wherein the        subject is a human.    -   52. An isolated polypeptide comprising amino acids 31-187 of        full length Lin28A or Lin28B polypeptide, wherein the isolated        polypeptide is less than 200 amino acids in length.    -   53. The isolated polypeptide of paragraph 52, wherein the        isolated polypeptide further comprises a deletion of at least        five amino acids between positions 121 to 138 of full length        Lin28A or Lin28B polypeptide.    -   54. The isolated polypeptide of paragraph 52 or 53, wherein the        isolated polypeptide comprises at least one modification        selected from a non-natural amino acid, a D-amino acid, a β        amino acid, a chemically modified amino acid, a modified amide        linkage, a tag amino acid sequence, and any combinations        thereof.    -   55. The isolated polypeptide of any of paragraphs 52-54, wherein        the polypeptide is useful for stem cell reprogramming.    -   56. The isolated polypeptide of any of paragraphs 52-55, wherein        the isolated polypeptide is selected from the group consisting        of        -   MHHHHHHENLYFQGSGAAEKAPEEAPPDAARAADEPQLLHGAGICKWFNVRMGFG            FLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTG            PGGVFCIGSERRPKGKNMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSI            NHMVASCPLKAQQGPSS (SEQ ID NO: 2);        -   MHHHHHHENLYFQGSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPP            VDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKG            GDRCYNCGGLDHHAKECKLPPQPKKCHFCQSINHMVASCPLKAQQGPSSQGK (SEQ ID            NO: 3);        -   MHHHHHHENLYFQGSGEEPEKLPGLAEDEPQVLHGTGHCKWFNVRMGFGFISMISR            EGNPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSPKGLESIRVTGPGGSPCLGS            ERRPKGKTLQKRKPKGDRWRRQDLLMDQMWTVREEESRMIPRCYNCGGLDHHAK            ECSLPPQPKKCHYCQSIMHMVANCPHKLAAQLPASS (SEQ ID NO: 4);        -   MHHHHHHENLYFQGSGAAEEAPEEAPEDAARAADEPQLLHGAGICKWFNVRMGFG            FLSMTARAGVALDPPVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTG            PGGVFCIGSERRPKGKSMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSIS            HMVASCPLKAQQGPSAQGK (SEQ ID NO: 5);        -   MHHHHHHENLYFQGSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPP            VDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKG            GDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLKAQQGPSAQGK (SEQ ID            NO: 6);        -   MHHHHHHENLYFQGSGEEPGKLPEPAEEESQVLRGTGHCKWFNVRMGFGFISMINR            EGSPLDIPVDVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGS            ERRPKGKTLQKRKPKGDRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCP            HKNVAQPPASSQGR (SEQ ID NO: 7);        -   MHHHHHHENLYFQGSGPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPV            DVFVHQSKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGSERRPKGGD            RCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKNVAQPPASSQGR (SEQ ID            NO: 8);        -   GSGAAEKAPEEAPPDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDP            PVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKG            KNMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSINHMVASCPLKAQQG PSS            (SEQ ID NO: 9);        -   GSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHME            GFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHH            AKECKLPPQPKKCHFCQSINHMVASCPLKAQQGPSSQGK (SEQ ID NO: 10);        -   GSGEEPEKLPGLAEDEPQVLHGTGHCKWFNVRMGFGFISMISREGNPLDIPVDVFVH            QSKLFMEGFRSLKEGEPVEFTFKKSPKGLESIRVTGPGGSPCLGSERRPKGKTLQKRK            PKGDRWRRQDLLMDQMWTVREEESRMIPRCYNCGGLDHHAKECSLPPQPKKCHYC            QSIMHMVANCPHKLAAQLPASS (SEQ ID NO: 11);        -   GSGAAEEAPEEAPEDAARAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDP            PVDVFVHQSKLHMEGFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKG            KSMQKRRSKGDRCYNCGGLDHHAKECKLPPQPKKCHFCQSISHMVASCPLKAQQGP            SAQGK (SEQ ID NO: 12);        -   GSGAADEPQLLHGAGICKWFNVRMGFGFLSMTARAGVALDPPVDVFVHQSKLHME            GFRSLKEGEAVEFTFKKSAKGLESIRVTGPGGVFCIGSERRPKGGDRCYNCGGLDHH            AKECKLPPQPKKCHFCQSISHMVASCPLKAQQGPSAQGK (SEQ ID NO: 13);        -   GSGEEPGKLPEPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQ            SKLFMEGFRSLKEGEPVEFTFKKSSKGLESIRVTGPGGSPCLGSERRPKGKTLQKRKP            KGDRCYNCGGLDHHAKECSLPPQPKKCHYCQSIMHMVANCPHKNVAQPPASSQGR (SEQ            ID NO: 14); and        -   GSGPAEEESQVLRGTGHCKWFNVRMGFGFISMINREGSPLDIPVDVFVHQSKLFMEG            FRSLKEGEPVEFTFKKSSKGLESIRVT (SEQ ID NO: 15).    -   57. A crystalline molecule or molecular complex comprising a        binding pocket of Lin28, wherein the Lin28 binding pocket is        defined by structure coordinates binding pocket of Tables 1-3        and said Tables 1-3 being optionally varied by a rmsd of less        than 1.5 Å or selected coordinates thereof.    -   58. The crystalline molecule or molecular complex of paragraph        57, wherein the Lin28 binding pocket comprises at least one        amino acid selected from the group consisting of D71, E105,        E106, F55, F73, F84, H75, K102, K45, K78, M51, R123, R50, R85,        S100, W45, W46, and any combinations from Table 1-3, or selected        coordinates thereof.    -   59. The crystalline molecule or molecular complex of paragraph        57 or 58, wherein the Lin28 binding pocket comprises the amino        acids D71, E106, F55, F84, H75, K102, K45, K78, M51, R50, R85,        S100, and W45; amino acids D71, E106, F55, F84, H75, K102, K78,        M51, R50, R85, S100, and W45; or amino acids D71, E105, F55,        F73, H75, K102, K78, M51, R123, R50, R85, S100, and W46 from        Tables 1-3 or selected coordinates thereof.    -   60. A crystal comprising Lin28 complexed with a pre-let-7,        wherein the crystal comprises structure coordinates of Tables        1-3.    -   61. A computer readable medium having Lin28 crystal structure        coordinates of Tables 1-3 stored thereon.    -   62. A computer based-method for analysis of interaction of        ligand with Lin28, the method comprising: providing Lin28        binding pocket of Tables 1-3, the Tables 1-3 being optionally        varied by a rmsd of less than 1.5A, or selected coordinates        thereof; providing a ligand structure to be fitted to the Lin28        binding pocket; and fitting the ligand structure to the Lin28        binding pocket, wherein the ligand structure is fitted to at        least one atom of an amino acid selected from the group        consisting of D71, D100, E105, F47, F55, F73, F84, G83, H75,        K102, K192, K45, K78, M51, N48, R122, R123, R50, R85, S100, V49,        W46, and any combinations thereof.    -   63. A screening assay for determining inhibitors of Lin28        activity, the method comprising contacting a polypeptide of any        of paragraphs 52-56 with a test compound and selecting the        compound that increases level of mature let-7 miRNA relative to        a control.    -   64. The method of paragraph 63, wherein the test compound is        selected from the group consisting of small organic or inorganic        molecules; peptides; proteins; peptide analogs and derivatives;        peptidomimetics; nucleic acids; nucleic acid analogs and        derivatives; an extract made from biological materials such as        bacteria, plants, fungi, or animal cells; animal tissues;        naturally occurring or synthetic compositions; and any        combinations thereof.    -   65. The method of any of paragraphs 63-64, wherein the test        compound has a molecular weight of less than 5000 Daltons (5        kD).    -   66. The method of any of paragraphs 63-65, wherein the test        compound is tested at a concentration in the range of about 0.1        nM to about 1000 mM.    -   67. The method of any of paragraphs 63-66, wherein the method is        a high-throughput screening method.    -   68. A compound selected by the method of any of paragraphs        63-67, and analogs, isomers, derivatives, and pharmaceutically        acceptable salts thereof.    -   69. A method of purifiying a Lin28 polypeptide, the method        comprising:        -   (i) expressing a Lin28 polypeptide or fragment thereof from            a vector in a cell;        -   (ii) purifying the Lin28 polypeptide using cation exchange            chromatography, wherin the cation exchange chromatography is            using a buffer comprising about 20 mM BisTris about pH 6.0,            about mM dithiothreitol (DTT), about 5% glycerol, and about            50 50 μM ZnCl₂, over 0.1-1M NaCl gradient.    -   70. The method of paragraph 69, comprising further purifying the        Lin28 polypeptide or fragment thereof of step (ii) with size        exclusion chromatography, wherin the size exclusion        chromatography is using a buffer comprising about 20 mM BisTris        about pH 6.0, about mM dithiothreitol (DTT), about 5% glycerol,        and about 50 50 μM ZnCl₂.    -   71. The method of paragraph 69 or 70, wherein the cell is        expressed in E. coli.    -   72. The method of any of paragraphs 69-71, wherein the expressed        Lin28 polypeptide comprises a His-tag, wherein the His-tag is        removed before purification by cation exchange chromatography.    -   73. The method of any of paragraphs 69-72, wherein the Lin28        polypeptide of fragment thereof is a polypeptide of any of        paragraphs 52-56.

Some Selected Definitions

Unless stated otherwise, or implicit from context, the following termsand phrases include the meanings provided below. Unless explicitlystated otherwise, or apparent from context, the terms and phrases belowdo not exclude the meaning that the term or phrase has acquired in theart to which it pertains. The definitions are provided to aid indescribing particular embodiments, and are not intended to limit theclaimed invention, because the scope of the invention is limited only bythe claims. Further, unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean ±1%.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease” , “reduced”, “reduction” , “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, ““reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased” ,“increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means a two standard deviation(2SD) below normal, or lower, concentration of the marker. The termrefers to statistical evidence that there is a difference. It is definedas the probability of making a decision to reject the null hypothesiswhen the null hypothesis is actually true. The decision is often madeusing the p-value.

The term “Lin28” as used herein is also referred to in the art asaliases LIN-28A, FLJ12457, ZCCHC1, Lin-28A, CSDD1 and Lin-28 homolog (C.elegans). Human Lin-28 is encoded by nucleic acid corresponding toGenBank Accession No: AF521099 (SEQ ID NO: 66) or RefSeq ID: NM_024674(SEQ ID NO: 67), and the human Lin-28 corresponds to protein sequencecorresponding to RefSeq ID:AAM77751 (SEQ ID NO: 68). Human Lin-28 has aconserved Cold Shock Domain (CSD) between residues 39-112, and two CCHCdomains; a type 1 CCHC domain between residues 137-154 and a type 2 CCHCdomain between resides 159-176. As used herein, the term

The term “Lin-28B” as used herein refers to a homologue of Lin28 and isalso known in the art as CSDD2, FLJ16517, or Lin-28.2. There are twoisoforms of , B, differing in their 5′ exons, have been reported, thelong isoform (Lin-28B-L also known as isoform 1 or identifier: Q6ZN17-1,corresponding to SEQ ID NO: 69 herein) which has two retroviral-typeCCHC zinc-finger motifs and a truncated cold-shock domain, and a shortisoform (Lin-28B-S, also known as isoform 2 or identifier: Q6ZN17-2,corresponding to SEQ ID NO: 70 herein) which preserves the tworetroviral-type CCHC zinc-finger motifs but contains a truncatedcold-shock domain (i.e. lacks 70 N-terminal amino acids as compared tothe Lin-28B-L isoform). Human Lin28B-L has a conserved Cold Shock Domain(CSD) between residues 29-102, and two CCHC domains; a type 1 CCHCdomain between residues 127-144 and a type 2 CCHC domain betweenresidues 149-166. Human Lin-28B-L is encoded by nucleic acidcorresponding to GenBank Accession No: AK131411 or RefSeq ID:NM_001004317 and the human Lin-28B corresponds to protein sequencecorresponding to RefSeq ID: NP_001004317.

The terms “microRNA” or “miRNA” or “miR” are used interchangeably hereinrefer to endogenous RNA molecules, which act as gene silencers toregulate the expression of protein-coding genes at thepost-transcriptional level. Endogenous microRNA are small RNAs naturallypresent in the genome which are capable of modulating the productiveutilization of mRNA. The term artificial microRNA includes any type ofRNA sequence, other than endogenous microRNA, which is capable ofmodulating the productive utilization of mRNA. MicroRNA sequences havebeen described in publications such as Lim, et al., Genes & Development,17, p. 991-1008 (2003), Lim et al Science 299, 1540 (2003), Lee andAmbros Science, 294, 862 (2001), Lau et al., Science 294, 858-861(2001), Lagos-Quintana et al, Current Biology, 12, 735-739 (2002), LagosQuintana et al, Science 294, 853-857 (2001), and Lagos-Quintana et al,RNA, 9, 175-179 (2003), which are incorporated by reference. MultiplemicroRNAs can also be incorporated into a precursor molecule.Furthermore, miRNA-like stem-loops can be expressed in cells as avehicle to deliver artificial miRNAs and short interfering RNAs (siRNAs)for the purpose of modulating the expression of endogenous genes throughthe miRNA and or RNAi pathways.

During miRNA maturation in animals, the primary transcript is firstprocessed to a stem-loop precursor and then the stem-loop is processedto yield a mature miRNA of about 22 nucleotides. These molecules candirect the cleavage of mRNA or they can interfere with productivetranslation of the mRNA, either of which results in reduced proteinaccumulation and hence the miRNAs are able to modulate gene expressionand related cellular activities. miRNAs are important in development anddifferentiation, and thus the altered expression of miRNAs could be usedto alter development and differentiation during tissue engineering andother applications . Furthermore, miRNA-like stem-loops can be expressedin cells as a vehicle to deliver artificial miRNAs and short interferingRNAs (siRNAs) for the purpose of modulating the expression of endogenousgenes through the miRNA and or RNAi pathways. Mimetics of miRNAsinclude, artificial miRNAs, and siRNAs are inefficient and are noteffective for many small RNA sequences.

The term “pri-miRNA” refers to a precursor microRNA molecule having amicroRNA sequence in the context of microRNA flanking sequences. Aprecursor microRNA, also referred to as large RNA precursors, arecomposed of any type of nucleic acid based molecule capable ofaccommodating the microRNA flanking sequences and the microRNA sequence.Examples of precursor microRNAs and the individual components of theprecursor (flanking sequences and microRNA sequence) are providedherein. The invention, however, is not limited to the examples provided.The invention is based, at least in part, on the discovery of animportant component of precursor microRNAs, that is, the microRNAflanking sequences. The nucleotide sequence of the precursor and itscomponents may vary widely. In one aspect a precursor microRNA moleculeis an isolated nucleic acid; including microRNA flanking sequences andhaving a stem-loop structure with a microRNA sequence incorporatedtherein.

A precursor microRNA molecule may be processed in vivo or in vitro toproduce a mature microRNA (miRNA). A precursor microRNA molecule isprocessed in a host cell by a ribonuclease enzyme or enzymes. Oneexample of a ribonuclease enzyme which processes precursor microRNAmolecules is the RNase II ribonuclease Dicer. [0075] The term“pre-miRNA” refers to the intermediate miRNA species from the processingof a pre-miRNA to a mature miRNA. Pre-miRNAs are produced from theprocessing of a pri-miRNA in the nucleus into a pre-miRNA. PremiRNAsundergo additional processing in the cytoplasm to form mature miRNA.Pre-miRNAs are approximately 70 nucleotides long, but can be less than70 nucleotides or more than 70 nucleotides.

The term “microRNA flanking sequence” as used herein refers tonucleotide sequences including microRNA processing elements. MicroRNAprocessing elements are the minimal nucleic acid sequences whichcontribute to the production of mature microRNA from precursor microRNA.Often these elements are located within a 40 nucleotide sequence thatflanks a microRNA stem-loop structure. In some instances the microRNAprocessing elements are found within a stretch of nucleotide sequencesof between 5 and 4,000 nucleotides in length that flank a microRNAstem-loop structure. Thus, in some embodiments the flanking sequencesare 5-4,000 nucleotides in length. As a result, the length of theprecursor molecule may be, in some instances at least about 150nucleotides or 270 nucleotides in length. The total length of theprecursor molecule, however, may be greater or less than these values.In other embodiments the minimal length of the microRNA flankingsequence is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 and anyinteger there between. In other embodiments the maximal length of themicroRNA flanking sequence is 2,000, 2,100, 2,200, 2,300, 2,400, 2,500,2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500,3,600, 3,700, 3,800, 3,900 4,000 and any integer there between.

The microRNA flanking sequences may be native microRNA flankingsequences or artificial microRNA flanking sequences. A native microRNAflanking sequence is a nucleotide sequence that is ordinarily associatedin naturally existing systems with microRNA sequences, i.e., thesesequences are found within the genomic sequences surrounding the minimalmicroRNA hairpin in vivo. Artificial microRNA flanking sequences arenucleotides sequences that are not found to be flanking to microRNAsequences in naturally existing systems. The artificial microRNAflanking sequences may be flanking sequences found naturally in thecontext of other microRNA sequences. Alternatively they may be composedof minimal microRNA processing elements which are found within naturallyoccurring flanking sequences and inserted into other random nucleic acidsequences that do not naturally occur as flanking sequences or onlypartially occur as natural flanking sequences. The microRNA flankingsequences within the precursor microRNA molecule may flank one or bothsides of the stemloop structure encompassing the microRNA sequence.Thus, one end (i.e., 5′) of the stem-loop structure may be adjacent to asingle flanking sequence and the other end (i.e., 3′) of the stem-loopstructure may not be adjacent to a flanking sequence. Preferredstructures have flanking sequences on both lo ends of the stem-loopstructure. The flanking sequences may be directly adjacent to one orboth ends of the stem-loop structure or may be connected to thestem-loop structure through a linker, additional nucleotides or othermolecules.

As used herein, the term “let-7” refers to the nucleic acid encoding thelet-7 miRNA family members and homologues and variants thereof includingconservative substitutions, additions, and deletions therein notadversely affecting the structure or function. Preferably, let-7 refersto the nucleic acid encoding let-7 from C. elegances (NCBI Accession No.AY390762), most preferably, let-7 refers to the nucleic acid encoding alet-7 family member from humans, including but not limited to, NCBIAccession Nos. AJ421724, AJ421725, AJ421726, AJ421727, AJ421728,AJ421729, AJ421730, AJ421731, AJ421732, and biologically active sequencevariants of let-7, including alleles, and in vitro generated derivativesof let-7 that demonstrate let-7 activity. Exemplary let-7 family membermiRNAs include, but are not limited to, Let-7, Let-7a-1, Let-7a-2,Let-7a-3, Let-7b, Let-7c, Let-7d, Let-7e, Let-7f-1, Let-7f-2, Let-7g,Let-7i, and miR-98.

The term “variant” as used herein refers to a peptide or nucleic acidthat differs from the naturally occurring polypeptide or nucleic acid byone or more amino acid or nucleic acid deletions, additions,substitutions or side-chain modifications, yet retains one or morespecific functions or biological activities of the naturally occurringmolecule. Amino acid substitutions include alterations in which an aminoacid is replaced with a different naturally-occurring or anon-conventional amino acid residue. Such substitutions may beclassified as “conservative”, in which case an amino acid residuecontained in a polypeptide is replaced with another naturally occurringamino acid of similar character either in relation to polarity, sidechain functionality or size. Substitutions encompassed by the presentinvention may also be “non conservative”, in which an amino acid residuewhich is present in a peptide is substituted with an amino acid havingdifferent properties, such as naturally-occurring amino acid from adifferent group (e.g., substituting a charged or hydrophobic amino; acidwith alanine), or alternatively, in which a naturally-occurring aminoacid is substituted with a nonconventional amino acid. In someembodiments amino acid substitutions are conservative. Also encompassedwithin the term variant when used with reference to a polynucleotide orpolypeptide, refers to a polynucleotide or polypeptide that can vary inprimary, secondary, or tertiary structure, as compared to a referencepolynucleotide or polypeptide, respectively (e.g., as compared to awild-type polynucleotide or polypeptide).

Variants can be naturally-occurring, synthetic, recombinant, orchemically modified polynucleotides or polypeptides isolated orgenerated using methods well known in the art. Variants can includeconservative or non-conservative amino acid changes, as described below.Polynucleotide changes can result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence. Variants can also include insertions,deletions or substitutions of amino acids, including insertions andsubstitutions of amino acids and other molecules) that do not normallyoccur in the peptide sequence that is the basis of the variant, forexample but not limited to insertion of ornithine which do not normallyoccur in human proteins. The term “conservative substitution,” whendescribing a polypeptide, refers to a change in the amino acidcomposition of the polypeptide that does not substantially alter thepolypeptide's activity. For example, a conservative substitution refersto substituting an amino acid residue for a different amino acid residuethat has similar chemical properties. Conservative amino acidsubstitutions include replacement of a leucine with an isoleucine orvaline, an aspartate with a glutamate, or a threonine with a serine.“Conservative amino acid substitutions” result from replacing one aminoacid with another having similar structural and/or chemical properties,such as the replacement of a leucine with an isoleucine or valine, anaspartate with a glutamate, or a threonine with a serine. Thus, a“conservative substitution” of a particular amino acid sequence refersto substitution of those amino acids that are not critical forpolypeptide activity or substitution of amino acids with other aminoacids having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or nonpolar, etc.) such that the substitutionof even critical amino acids does not reduce the activity of thepeptide, (i.e. the ability of the peptide to penetrate the BBB).Conservative substitution tables providing functionally similar aminoacids are well known in the art. For example, the following six groupseach contain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid(D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine(R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine(V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). (See alsoCreighton, Proteins, W. H. Freeman and Company (1984).) In someembodiments, individual substitutions, deletions or additions thatalter, add or delete a single amino acid or a small percentage of aminoacids can also be considered “conservative substitutions” is the changedoes not reduce the activity of the peptide (i.e. the ability of aLin-28 polypeptide to process the maturation of miRNA). Insertions ordeletions are typically in the range of about 1 to 5 amino acids. Thechoice of conservative amino acids may be selected based on the locationof the amino acid to be substituted in the peptide, for example if theamino acid is on the exterior of the peptide and expose to solvents, oron the interior and not exposed to solvents.

The term “derivative” as used herein refers to peptides which have beenchemically modified, for example but not limited to by techniques suchas ubiquitination, labeling, pegylation (derivatization withpolyethylene glycol) or addition of other molecules. A molecule also a“derivative” of another molecule when it contains additional chemicalmoieties not normally a part of the molecule. Such moieties can improvethe molecule's solubility, absorption, biological half-life, etc. Themoieties can alternatively decrease the toxicity of the molecule,eliminate or attenuate any undesirable side effect of the molecule, etc.Moieties capable of mediating such effects are disclosed in Remington'sPharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., MackPubl.,Eastern, Pa. (1990).

The term “functional” when used in conjunction with “derivative” or“variant” refers to a molecule such as a protein which possess abiological activity (either functional or structural) that issubstantially similar to a biological activity of the entity or moleculeis a functional derivative or functional variant thereof The termfunctional derivative is intended to include the fragments, analogues orchemical derivatives of a molecule.

A molecule is said to be “substantially similar” to another molecule ifboth molecules have substantially similar structures or if bothmolecules possess a similar biological activity, for example if bothmolecules are able to deliver a target antigen to the cytosol of a cellin the absence of PA and without being fused to the target antigen.Thus, provided that two molecules possess a similar activity, areconsidered variants and are encompassed for use as disclosed herein,even if the structure of one of the molecules not found in the other, orif the sequence of amino acid residues is not identical. Thus, providedthat two molecules possess a similar biological activity, they areconsidered variants as that term is used herein even if the structure ofone of the molecules not found in the other, or if the sequence of aminoacid residues is not identical.

As used herein, the term “non-conservative” refers to substituting anamino acid residue for a different amino acid residue that has differentchemical properties. The nonconservative substitutions include, but arenot limited to aspartic acid (D) being replaced with glycine (G);asparagine (N) being replaced with lysine (K); or alanine (A) beingreplaced with arginine (R).

The term “disease” or “disorder” is used interchangeably herein, refersto any alternation in state of the body or of some of the organs,interrupting or disturbing the performance of the functions and/orcausing symptoms such as discomfort, dysfunction, distress, or evendeath to the person afflicted or those in contact with a person. Adisease or disorder can also related to a distemper, ailing, ailment,malady, disorder, sickness, illness, complaint, inderdisposion,affection.

The term “malignancy” and “cancer” are used interchangeably herein,refers to diseases that are characterized by uncontrolled, abnormalgrowth of cells. Cancer cells can spread locally or through thebloodstream and lymphatic system to other parts of the body. The term“malignancy” or “cancer” are used interchangeably herein and refers toany disease of an organ or tissue in mammals characterized by poorlycontrolled or uncontrolled multiplication of normal or abnormal cells inthat tissue and its effect on the body as a whole. Cancer diseaseswithin the scope of the definition comprise benign neoplasms,dysplasias, hyperplasias as well as neoplasms showing metastatic growthor any other transformations like e.g. leukoplakias which often precedea breakout of cancer.

The term “tumor” or “tumor cell” are used interchangeably herein, refersto the tissue mass or tissue type of cell that is undergoing abnormalproliferation.

A “cancer cell” refers to a cancerous, pre-cancerous or transformedcell, either in vivo, ex vivo, and in tissue culture, that hasspontaneous or induced phenotypic changes that do not necessarilyinvolve the uptake of new genetic material. Although transformation canarise from infection with a transforming virus and incorporation of newgenomic nucleic acid, or uptake of exogenous nucleic acid, it can alsoarise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation/cancer is associated with,e.g., morphological changes, immortalization of cells, aberrant growthcontrol, foci formation, anchorage dependence, proliferation,malignancy, contact inhibition and density limitation of growth, growthfactor or serum dependence, tumor specific markers levels, invasiveness,tumor growth or suppression in suitable animal hosts such as nude mice,and the like, in vitro, in vivo, and ex vivo (see Example VII) (see alsoFreshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed.1994)).

A “sarcoma” refers to a type of cancer cell that is derived fromconnective tissue, e.g., bone (osteosarcoma) cartilage (chondrosarcoma),muscle (rhabdomyosarcoma or rhabdosarcoma), fat cells (liposarcoma),lymphoid tissue (lymphosarcoma), collagen-producing fibroblasts(fibrosarcoma). Sarcomas may be induced by infection with certainviruses, e.g., Kaposi's sarcoma, Rous sarcoma virus, etc.

The term “biological sample” as used herein may mean a sample ofbiological tissue or fluid that comprises nucleic acids. Such samplesinclude, but are not limited to, tissue isolated from animals.Biological samples may also include sections of tissues such as biopsyand autopsy samples, frozen sections taken for histologic purposes,blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin.Biological samples also include explants and primary and/or transformedcell cultures derived from patient tissues. A biological sample may beprovided by removing a sample of cells from an animal, but can also beaccomplished by using previously isolated cells (e.g., isolated byanother person, at another time, and/or for another purpose), or byperforming the methods of the invention in vivo. Archival tissues, suchas those having treatment or outcome history may also be used. As usedherein, the term “biological sample” also refers to a cell or populationof cells or a quantity of tissue or fluid from a subject. Most often,the sample has been removed from a subject, but the term “biologicalsample” can also refer to cells or tissue analyzed in vivo, i.e. withoutremoval from the subject. Often, a “biological sample” will containcells from the animal, but the term can also refer to non-cellularbiological material, such as non-cellular fractions of blood, saliva, orurine, that can be used to measure gene expression levels. Biologicalsamples include, but are not limited to, tissue biopsies, scrapes (e.g.buccal scrapes), whole blood, plasma, serum, urine, saliva, cellculture, or cerebrospinal fluid. Biological samples also include tissuebiopsies, cell culture. A biological sample or tissue sample can refersto a sample of tissue or fluid isolated from an individual, includingbut not limited to, for example, blood, plasma, serum, tumor biopsy,urine, stool, sputum, spinal fluid, pleural fluid, nipple aspirates,lymph fluid, the external sections of the skin, respiratory, intestinal,and genitourinary tracts, tears, saliva, milk, cells (including but notlimited to blood cells), tumors, organs, and also samples of in vitrocell culture constituent. In some embodiments, the sample is from aresection, bronchoscopic biopsy, or core needle biopsy of a primary ormetastatic tumor, or a cellblock from pleural fluid. In addition, fineneedle aspirate samples are used. Samples may be eitherparaffin-embedded or frozen tissue. The sample can be obtained byremoving a sample of cells from a subject, but can also be accomplishedby using previously isolated cells (e.g. isolated by another person), orby performing the methods of the invention in vivo. Biological samplealso refers to a sample of tissue or fluid isolated from an individual,including but not limited to, for example, blood, plasma, serum, tumorbiopsy, urine, stool, sputum, spinal fluid, pleural fluid, nippleaspirates, lymph fluid, the external sections of the skin, respiratory,intestinal, and genitourinary tracts, tears, saliva, milk, cells(including but not limited to blood cells), tumors, organs, and alsosamples of in vitro cell culture constituent. In some embodiments, thebiological samples can be prepared, for example biological samples maybe fresh, fixed, frozen, or embedded in paraffin.

The term “tissue” is intended to include intact cells, blood, bloodpreparations such as plasma and serum, bones, joints, muscles, smoothmuscles, and organs.

As used herein, the term “treating” includes reducing or alleviating atleast one adverse effect or symptom of a condition, disease or disorderassociated with inappropriate proliferation, for example cancer. As usedherein, the term treating is used to refer to the reduction of a symptomand/or a biochemical marker of in appropriate proliferation, for examplea reduction in at least one biochemical marker of cancer by at least10%. For example but are not limited to, a reduction in a biochemicalmarker of cancer, for example a reduction in, as an illustrative exampleonly, at least one of the following biomarkers; CD44, telomerase, TGF-α,TGF-β, erbB-2, erbB-3, MUC1, MUC2, CK20, PSA, CA125, FOBT, by 10%, or areduction in the rate of proliferation of the cancer cells by 10%, wouldbe considered effective treatments by the methods as disclosed herein.As alternative examples, a reduction in a symptom of cancer, forexample, a slowing of the rate of growth of the cancer by 10% or acessation of the increase in tumor size, or a reduction in the size of atumor by 10% or a reduction in the tumor spread (i.e. tumor metastasis)by 10% would also be considered as affective treatments by the methodsas disclosed herein.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various embodiments hereindescribed and illustrated may be further modified to incorporatefeatures shown in any of the other embodiments disclosed herein.

The following examples illustrate some embodiments and aspects of theinvention. It will be apparent to those skilled in the relevant art thatvarious modifications, additions, substitutions, and the like can beperformed without altering the spirit or scope of the invention, andsuch modifications and variations are encompassed within the scope ofthe invention as defined in the claims which follow. The followingexamples do not in any way limit the invention.

EXAMPLES Example 1: Molecular Basis for Interaction of let-7 microRNAswith Lin28 Materials and Methods

Constructs: All Lin28 crystallization constructs were derived from mouseLin28a (NP_665832). Expression constructs are in pETDuet-1 (Novagen),with an N-terminal hexahistidine tag (SEQ ID NO: 71) followed by a TEVcleavage site. For NMR studies and electrophoretic mobility shiftassays, Lin28 (residues 16-184) contained the wild-type linker sequence,unless otherwise noted. For crystallization a similar construct (35-187)with nine internally deleted residues in the linker (49) was used (FIG.2B). Isolated CSD construct contains residues 16-126 and CCHCx2construct contains residues 135-184.

Protein purification and complex preparation: Lin28 constructs derivedfrom mouse Lin28a were purified after overexpression in E. coli, usingNickel affinity, cation exchange, and size exclusion chromatography.Specifically, Lin28 constructs were overexpressed in E. coli strainBL21(DE3) Rosetta pLysS. After initial affinity chromatography stepusing Ni-NTA beads (Qiagen), His-tags were removed by incubating withrecombinant TEV protease. Cation exchange chromatography (HiTrap S, GEHealthcare) was performed using a buffer containing 20 mM BisTris pH6.0, 5 mM dithiothreitol (DTT), 5% glycerol, and 50 μM ZnCl₂, over0.1-1M NaCl gradient. Further purification was accomplished by sizeexclusion chromatography (Superdex 200, GE Healthcare) in the samebuffer. Complexes with RNA oligonucleotides were prepared for NMRstudies and crystallization trials, by mixing at 1:1.2 (protein:RNA)molar ratio, and free RNA was removed by another size-exclusionchromatography step.

Electrophoretic Mobility Shift Assay: For preE probes, RNAoligonucleotides were synthesized (IDT), and full pre-miR probes werepurified by PAGE after in vitro transcription followed by doubleribozyme cleavage, as detailed in (Walker et al., 2003). RNAs wereradiolabeled with ATI:[γ-³²P] using T4 polynucleotide kinase, incubatedwith protein in a buffer containing 20 mM Tris 7.5, 100 mM NaCl, 10 mMDTT, 50 μM ZnCl₂, 15 μg/μL yeast tRNA, and 1U/μL RNAse inhibitor.

Equilibrium Sedimentation: Complexes containing indicated protein andRNA constructs were purified as described above. Three concentrations(Absorbance₂₈₀=0.2, 0.4, 0.6) were measured for each complex. Data wascollected on a Beckman Optima XL-A ultracentrifuge at 4 speeds (15, 18,21, 24K RPM) and analyzed by fitting to a single-species model usingOrigin. Partial specific volumes for each complex was calculated byusing NucProt (Voss and Gerstein, 2005).

NMR spectroscopy: All NMR samples were prepared as 0.5 mM Lin28 (16-184)and preE_(M)-let-7d complex, in a buffer containing 20 mM BisTris pH7.0, 100 mM NaCl, 5% glycerol, 5 mM dithiothreitol, 50 μM ZnCl₂, and0.2% sodium azide. Backbone distance restraints were obtained usinguniform ¹⁵N, ¹³C-labeled protein in complex with unlabeled RNA, from 3D¹⁵N-edited NOESY (mixing time=120 ms). To measure ¹⁵N R₁ and R₂, asample containing ¹⁵N, ¹³C and 85% ²H-labeled protein combined withunlabeled RNA was used with standard pulse schemes (Kay et al., 1989).Secondary chemical shifts were calculated by the comparing the recordedchemical shift to sequence adjusted random coil chemical shift(Schwarzinger et al., 2001). Sequence-specific chemical shifts forbackbone atoms were determined for 157 residues (out of 166 total,including 13 prolines), using the TROSY versions of HNCA, HN(CO)CA,HNCACB, HN(CO)CACB, HNCO, and HN(CA)CO, using a ¹⁵N, ¹³C and 85%²H-labeled protein combined with unlabeled RNA. Experiments wereconducted at 30° C. on Bruker spectrometers equipped with cryogenicprobes, operating at 1H frequencies of 600 MHz (sequence assignment andrelaxation experiments) or 750 MHz (NOESYs).

All spectra were processed and analyzed with NMRPipe (Delaglio et al.,1995) and CCPnmr Analysis (Vranken et al., 2005).

Crystallography: Crystals of all three complexes were produced by vapordiffusion, using the hanging drop method. Concentrated complexes (10mg/mL) were mixed 1 μL:1 μL with reservoir solution, and crystals grewovernight. Reservoir solution contained 0.6M NaH₂PO₄, 1.4M K₂HPO₄ and 5%glycerol for preE_(M)-let-7d and preE_(M)-let-7f-1 complexes; forpreE_(M)-let-7g, it contained 0.1M Tris pH 8.0, 32% w/v PEG 4000, and0.2M Sodium Acetate. Crystals were harvested with mother liquorsupplemented with 20% glycerol and frozen in liquid nitrogen.Diffraction data was indexed and scaled using XDS (Kabsch, 2010) andSCALA (Evans, 2006) in a workflow provided by autoPROC (Vonrhein et al.,2011). Experimental phases were obtained for Lin28:pre-let-7d complex byanomalous scattering from zinc atoms (SAD), using HKL2MAP (Schneider andSheldrick, 2002) and AutoSol (Terwilliger et al., 2009). The structuresof Lin28: preE_(M)-let-7f-1 and Lin28: preEM-let-7g were solved bymolecular replacement with Lin28: preEM-let-7d as search model usingPhaser (McCoy et al., 2007). Density modification and NCS averaging over6 or 2 copies were performed with PHENIX (Adams et al., 2010) to obtainelectron density maps for model building with COOT (Emsley and Cowtan,2004) and PHENIX was used for further refinement. Final rounds ofrefinement were carried out using BUSTER with local structure similarityrestraints (LSSR) and TLS (Bricogne et al., 2011).

Dicer in vitro processing assay: Dicer expression construct (Addgeneplasmid 19873) and purification are described as in (Landthaler et al.,2008), and radiolabeled pre-miR constructs were prepared similarly toEMSA probes. Dicer assays were carried out as described in (De andMacrae, 2011), using a buffer containing 20 mM Tris 7.5, 5% glycerol,3.2 mM MgCl₂, 5 mM DTT, 50 mM NaCl, and 100 μM ZnCl₂.

MicroRNA in vivo processing assay: Ability of Lin28 constructs to blocklet-7 processing in cells was compared as outlined in (Viswanathan etal., 2008). Briefly, pri-let-7g was co-transfected with FLAG-taggedLin28 constructs (25 ng unless otherwise noted) or vector control into293T cells (12 well) using lipofectamine. Total RNA was isolated usingTriZol reagent, treated with DNAse I, and quantitative RT-PCR was usedwith miRNA-specific stem-loop primers as previously described (Wan etal., 2010). Relative levels of mature miRNAs were analyzed by 44Ctmethod, and normalized by U6 snRNA levels.:

Accession numbers: Coordinates and structure factors for the structuresof Lin28:preE_(M)-let-7d, Lin28:preE_(M)-let-7f-1, andLin28:preE_(M)-let-7g complexes are disclosed in Tables 1-3 and havebeen deposited with the Protein Data Bank under accession codes 3TRZ,3TS0, and 3TS2.

Results and Discussion

Two discrete binding sites in pre-let-7 for Lin28 binding: As a firststep to understanding how pre-let-7 is recognized by Lin28, theinventors tested a series of deletions in pre-let-7d for binding to theprotein. Pre-let-7d has a relatively high affinity for Lin28 both invivo and in vitro (Hagan et al., 2009,Heo et al., 2009,Newman et al.,2008), and secondary structure predictions indicate that it has the moststable preE-stem among mouse pre-let-7s, without interrupting bulges(Markham and Zuker, 2005). The inventors focused their analysis on thepreE, as mutagenesis studies had indicated its importance in directassociation with Lin28 (Heo et al., 2009, Newman et al., 2008,Piskounova et al., 2008, Rybak et al., 2008). Innetos discovered that anisolated preE segment, containing none of the mature-region nucleotides,can bind Lin28 and that two distinct regions are critical for binding toLin28, thereby defining a minimal preE-let-7d (preE_(M)-let-7d)sufficient for high-affinity binding (FIG. 1C, 8B-C). The first requiredregion includes the preE-stem and the preE-loop; truncating the stemreduces binding. The other is the GGAG motif, which occurs at the 3′ endof the preE bulge. Although overall preE sequence conservation is low,even within the preE stem and loop, the GGAG tetranucleotide element iswell conserved throughout the let-7 family (FIG. 8A). Inventors' mappingresults indicated that the GGAG element provides an independent bindingsite, as deleting the neighboring nucleotides, thereby altering thedistance to the CSD binding site, does not abolish Lin28 binding.Without wishing to be bound by a theory, the presence of two independentbinding sites can explain how diverse preE-let-7s containing variablelinker sequences can all bind Lin28 with high specificity and affinity.

Domains of Lin28 tethered to each other are sufficient for inhibitinglet-7 processing: Lin28 has two folded regions, CSD and CCHCx2,connected by a positively charged linker of ˜15 amino acids, withextensions of ˜30 residues at both the amino and carboxy termini.Mutagenesis studies have implicated both folded domains in repression oflet-7 (Heo et al., 2009, Piskounova et al., 2008). The region C-terminalto the CCHCx2 domain also promotes translation of certain mRNA targets(Jin et al., 2011, Peng et al., 2011, Qiu et al., 2010). Using limitedproteolysis and electrophoretic mobility shift assay (EMSA), theinventors analyzed a series of truncation constructs of Lin28 toidentify the essential region for interaction with preE-let-7. Both theN- and C-terminal regions can be removed without affecting affinity forRNA, but removal of either the CSD or the CCHCx2 abolishes high-affinitypreE-let-7 binding (FIG. 9A).

The inventors used NMR spectroscopy to study the dynamics ofLin28:preE_(M)-let-7d complexes in more detail (FIG. 2A). They measuredlongitudinal (R1) and transverse (R2) relaxation rates to probe backbonedynamics. The R2/R1 ratio, which is a measure of correlation time, is anindicator of tumbling rate in solution. This ratio is similar for thefolded domains but much lower for the terminal segments and theintervening linker, indicating more rapid motion in those regions. Thisindicates that the linker sequence lacks secondary structure, aninference consistent with absence of inter-residue backbone NOEcrosspeaks in ¹⁵N-NOESY (FIG. 9B). Comparing the Cα, Cβ, C′ chemicalshifts to random coil chemical shifts also indicates that the linkerregion lacks secondary structure (FIG. 9C). Deletion of up to 9 aminoacids in the linker region supports binding to preE-let-7d orpreE-let-7f-1, although further deletion prevents complex formation(FIG. 2B-C, 9D). This indicates that a Lin28 fragment (31-187) with N-and C-terminal truncations and a 9-residue linker deletion (Lin28ΔΔ), issufficient for binding to preE-let-7 in vitro.

To test whether Lin28ΔΔ can inhibit let-7 processing in cells, theinventors compared the intracellular levels of processed mature let-7gwhen pri-let-7g is co-transfected with different Lin28 truncationconstructs. Relative to vector alone, Lin28ΔΔ significantly reduces thelevel of mature let-7g, although not as much as the full-length Lin28construct, probably due to slightly lower affinity (FIG. 2C-D).Processing of pri-miR-122 or pri-miR-16 is not inhibited by either Lin28construct (FIG. 9E). Ectopically expressed Lin28 levels are similar tothe endogenous levels observed in P19 cells and also among all Lin28constructs (FIG. 2E-F). The Lin2844 construct is therefore comparable tothe full-length protein in its ability to inhibit processing in vivo aswell as to bind let-7 precursors in vitro.

High resolution crystal structures of Lin28 with let-7 microRNA: Theinventors determined crystal structures of Lin2844 in complex withpreEM-let-75 derived from let-7d, let-7f-1, and let-7g, at resolutions2.9 Å, 2.8 Å, and 2.0Å, respectively, from three different crystal forms(FIGS. 3, 10A). They used single-wavelength anomalous dispersion (SAD),with the bound zinc atoms as the anomalous scatterers, to determine thestructure of the Lin28ΔΔ:preE_(M)-let-7d complex; we determined theother structures by molecular replacement. While the overallarchitectures of the three complexes are similar (Lin28 Ca RMSD<1.3 Å),there are several local differences due to divergent RNA sequences (FIG.10B-C, and see CSD and CCHC sections below).

The structures reveal that the CSD and CCHCx2 domains of Lin28 interactwith two distinct single-stranded regions of the RNA fragment (FIG. 3A).The preE-loop encircles a protrusion of the CSD as a necktie would wraparound a collar, with the extensive contacts around the circle madepossible by the presence of the preE-stem, which functions as thenecktie's knot. The CCHC zinc knuckles interact with the GGAG motif atthe 3′ end, and several sequence-specific interactions shape the singlestranded segment around the knuckles to introduce a distinctive kink inthe RNA backbone. Positively charged surfaces on both domains interactwith RNA throughout the complex (FIG. 3B).

The shortened linker between CSD and CCHCx2 is the most variable regionamong the different complexes (FIG. 10D). In all three crystal forms,the inventors discovered a domain swap in which the Lin28 CSD interactswith the loop of one RNA molecule and the CCHCx2 interacts with the GGAGof a second RNA (FIG. 3C). That is, each Lin28 monomer in the crystalinteracts with distinct elements of two separate preEM-let-7d molecules.In sedimentation equilibrium ultracentrifugation experiments under morephysiological conditions, we observe only monomeric complexes ofLin28:preE-let-7d, with or without internal deletions in the Lin28linker (FIG. 10E). An unswapped complex conformation can be modeled witha small rearrangement of the C-terminal extension of the CSD (residues112-121) and a rotation of the 7-residue linker to span the 18-30 Ådistance between CSD and CCHCx2 on the same RNA (FIG. 3A). Moreover, thelonger, 16-residue linker in wildtype Lin28 would accommodate evenlonger RNA substrates, including pre-let-7d without internal deletions.The monomeric model is also consistent with our observation that highaffinity RNA binding by Lin28 requires both Lin28-binding sites on thesame molecule (FIG. 1C). As all biochemical evidence points to amonomeric complex in solution, the description is restricted to a 1:1complex, with CSD and CCHCx2 bound in cis to a single RNA.

Specific binding of preE-let-7 with CSD: A detailed analysis of thecontacts between the CSD and the preE-let-7 stem-loops suggests thatspecificity relies on both the sequence and the conformation of the RNA.Most of the direct contacts lie in a ≥9-nucleotide segment that includesthe preE-loop (FIG. 4, 11A-B). As the loop wraps around the CSD, thebases project and make a number of π-stacking interactions with aromaticside chains. Complementary to the Velcro-like effects of the hydrophobicinteractions, hydrogen bonding and steric exclusion create nucleotidepreferences and enhance specificity. From inspection of the bindingpocket of each nucleotide, we can imagine an ideal RNA substrate for theCSD of Lin28. To simplify the discussion, the inventors define themiddle position of preE-let-7d that docks into the pocket lined by Phe73and Lys102 as the “center”, or position 0. Purines are preferred atpositions 0 and −1, near the tip of the loop so that the bulky bases canreach the protein. Position 1, on the other hand, is limited to apyrimidine, as Lys45 and Asp71 impose steric hindrance. A deeper pocketat position −3 makes a purine more favorable, because a larger ring isnecessary to stack over Phe84 (in d and f-1) and also to make favorablecontacts with the Lin28 backbone (in all three). The hydrogen bondingnetworks around −3, −1 and 0 are specific for G, G and A, respectively.

The inventors evaluated the effect of several point mutations in theco-crystallized preEs at positions where specific interactions areobserved in the structures (FIG. 4C, 11C). Most of the mutant probeshave lower affinity for Lin28 than wild-type. Although Gua is stronglypreferred over Ade at positions −3 and −1, substitution of Ade₀ with aGua is not as deleterious. Ade replaces Gua⁻³ in the Lin28:let-7gcomplex, and as a result some favorable hydrogen bonds are absent incomparison to other structures. Due to the small size of the pocket, apyrimidine is strongly preferred at position 1. Some of the previouslyreported mutations of preE-let-7g include a transversion (purine topyrimidine) at position 0 (Newman et al., 2008) and changes in thepreE-stem that disrupt base pairing (Piskounova et al., 2008). While thestudies described herein in focused on mouse Lin28a, the observedeffects of preE_(M) point mutations on complex stability are equivalentfor human Lin28a and Lin28b (FIG. 11C).

Comparing the structure of Lin28 bound to the divergent preE-let-7g withthose of the preE-let-7d and -7f-1 complexes illustrates how the CSDaccommodates variability in substrate RNAs. The short preE-loops inlet-7d and -f-1 require that base pairs be broken to fit around the CSD.In order to tighten the longer loop in preE-let-7g (FIG. 4B), Arg50moves in to mimic a base, pairing with Cyt⁻⁵ and stacking against Ade₅.The other extra bases have π-stacking interactions: two with the sidechains of Arg122 and Arg123 at the amino-terminal end of theinter-domain linker, and Ade₂ and Cyt₃ with each other. A closed RNAloop appears to be important to maintain full contact with the CSD,perhaps explaining the more extensive interactions here than in otherCSD:RNA complex structures (Frazão et al., 2006,Max et al., 2006, 2007)(FIG. 11D).

Interactions of zinc knuckles with preE-let-7: The CCHC knucklesmaximize favorable interactions with a small number of nucleotides bymaking many contacts with the bases (FIG. 5A-C). The intimateinteraction between GGAG and CCHCx2 produces a distinctive kink in theRNA backbone. Most of the protein atoms participating in the extensivehydrogen bonding network lie in relatively rigid regions of the protein,such as adjacent to zinc-coordinating residues or in a proline-richlinker, thereby imposing a specific, rigid conformation on the 3′ end ofthe RNA (FIG. 5C, 12A-B). Ring stacking and hydrophobic interactionswith side chains of the CCHCx2 further stabilize the particularconformation by aligning the bases. One of the key residues is Y140,which establishes the kinked conformation by sandwiching between thelast two bases (AG) and interacting with H162, which braces the first(G). Although the adenine base does not have as many polar contacts withLin28, it packs closely against the first Gua and makes a hydrogen bondthat assists in bending the RNA backbone. The resulting conformation ofthe ssRNA resembles that of the so-called “K-turn”, which oftenparticipates in specific protein-RNA interactions (Klein et al., 2001).

The CCHCx2 regions from all our structures align well with each other,except for slight differences, due to crystal contacts, in one of thetwo non-crystallographic copies of preE_(M)-let-7g (FIG. 12C-D). Whencompared with the conformation seen in the solution structure of anisolated Lin28 zinc-knuckle fragment (PDB 2CQF), however, there is alarge rearrangement of the inter-knuckle joint in Lin28 (FIG. 5D).Therefore, association of CCHCx2 with GGAG imposes specificconformational constraints on both the RNA and the protein; thisreciprocal effect may be functionally important for regulation.

Two NMR structures of CCHC motifs from HIV NCp1 have been determinedpreviously, in which the knuckles bind a tetraloop of sequence GGAG orGGUG in two stem loops (SL2 and SL3) of the _(W)-site (Amarasinghe etal., 2000,De Guzman et al., 1998). The conformation of the GGAG motif incomplex with Lin28 is very different from its conformation in complexwith HIV NCp1, indicating that the conformation we observe is specificto Lin28 (FIG. 12E-F).

Lin28 interactions with full-length pre-let-7: To test their conclusionsfrom the model provided by the crystal structures, and to verify thatthe truncations and deletions they had made for crystallization did notaffect specificity, the inventors generated mutant forms of full-lengthLin28 and pre-let-7g. Alteration of the key binding sites of CSD (nearposition 0) or CCHCx2 (GGAG) in pre-let-7g reduces affinity, consistentwith the mutagenesis studies with preE fragments (FIG. 6A, 13A). Inaddition, mutation of RNA-contacting residues in CSD and CCHCx2 alsointerferes with complex formation, especially when aromatic side chainsare replaced with Ala (FIG. 6B, 13B). We then conducted binding assaysusing combinations of protein and RNA mutants (FIG. 6C, 3C). The D71side chain, which is near nucleotide position 1, limits the size of thepocket and restricts it to pyrimidine rings. Presumably due to theadditional free space provided by a glycine, a D71G mutant no longerdiscriminates against a purine at position 1 (FIG. 6C, D71G block).

The bipartite character of the Lin28:let-7 interactions implies that oneshould observe strong synergy when combining a mutation in one of thetwo let-7 interaction sites with a mutation in the Lin28 domain thatrecognizes the other let-7 interaction site. Indeed, a CSD mutation(F73A) has much greater effect on binding with RNA bearing a mutation inthe GGAG motif (to GGAU or deletion) than it does on binding with RNAbearing a preE-loop mutation near the CSD binding site (FIG. 6C, F73Ablock). Similarly, for binding with a mutated CCHCx2 (Y140A), GGAGmutations are not as detrimental as a CSD binding-site mutation (FIG.6C, Y140A block). The inventors also tested binding of individualdomains of Lin28 to various pre-let-7g mutants (FIG. 6C, CSD and CCHCx2blocks). Neither isolated domain binds to let-7 as specifically ortightly as does full-length Lin28. Nevertheless, RNA mutations at eachbinding site affect only the affinity of the corresponding domain,consistent with the model presented herein. In summary, the results ofall these mutational studies are all consistent with the conclusion thatLin28 binds full-length pre-let-7 in the same way as does the truncatedform present in the crystals described herein.

The GGAG motif is conserved among let-7s not only in its sequence butalso in its proximal position with respect to the Dicer site in thecontext of the full pre-let-7 molecule. The last G is 4 bases from theDicer cleavage site on the 3′ strand, and only 2 bases from the positionat which complementarity to the mature strand begins. Using previouslydetermined structures of Dicer and the proposed location of the cut site(Du et al., 2008,Macrae et al., 2006), the inventors have modeled how aLin28:pre-let-7 complex would interact with Dicer (FIG. 13D). Becausetheir binding sites on RNA are close together and because Lin28 bendsthe RNA backbone, Lin28, especially its CCHCx2, can hinder Dicerdirectly. To test whether binding of Lin28 with pre-let-7g is sufficientto inhibit Dicer processing, the inventors used different mutants in anin vitro Dicer assay (FIG. 6D). The mutations that disrupt associationbetween Lin28 and pre-let-7 lead to increased Dicer cleavage, comparedwith wildtype control. The data presented herein are thus consistentwith a direct effect of Lin28 on Dicer processing of pre-let-7.

The inventors also tested the effect of the described mutations on invivo processing of let-7 (FIG. 6E-G). Mutations that affect CSD bindingde-repress processing of pri-let-7g only modestly, perhaps because thepresence of other cellular factors partially compensate for the affinitychange (<10 fold). Altering the CCHCx2:GGAG interaction—by changes inRNA or protein—is more detrimental to Lin28 activity. Levels of maturelet-7 in our in vivo assay depend on both complex formation betweenLin28 and let-7 precursors and downstream effects of Lin28, such ashindering Drosha and Dicer while recruiting TUTase. The results indicatethat although both CSD and CCHCx2 contribute to affinity and specificityfor let-7 precursors, the CCHCx2:GGAG interaction is more critical forthe effector function of Lin28.

Deciphering Lin28 specificity: The structural and biochemical studiespresented here reveal how Lin28 recognizes let-7 precursors and allow usto postulate how Lin28 might bind diverse pre-let-7s. The inventors havediscovered a preferred sequence consensus for CSD binding: NGNGA₀YNNN(Y=pyrimidine; N=any base). The sequences and distances between the CSDbinding site and the CCHCx2-binding GGAG motif are variable, but the twosites can be identified in many of the preE-let-7 sequences (FIG. 14A).Without wishing to be bound by a theory, in cases where no significantpreE-stem structure is predicted (e.g., in let-7a-2 or let-7c-1), thenearby mature region with its stable double-stranded helix can aid inclosing the loop around the CSD. Loss of one or a few favorableinteractions in other preE-let-7s might not completely exclude the RNAsfrom binding to Lin28, but rather result in differences in affinity thatcould affect the sensitivity of particular let-7s to Lin28 regulation invivo. Indeed, understanding Lin28 specificity from preE-let-7d andpreE-let-7f-1 allowed the inventors to crystallize the preE-let-7gcomplex, which binds to Lin28 in an energetically less stableconformation (FIG. 14B-C).

The sequence of the linker between CSD and CCHCx2 has a strong netpositive charge, probably to interact with the negatively charged RNAsugar-phosphate backbone, or to compensate for any unpaired bases, asseen in the case of preE-let-7g complex. Evolutionary conservation ofthe electrostatic property suggests that the linker does play some role,even though its sequence is not crucial for binding specificity. Thelength of the linker varies in some organisms, and shorter linkers occurin those with only one copy of let-7 containing a shorter preE sequence.Longer, more flexible linkers might have evolved in higher eukaryotes torecognize longer and divergent let-7 precursors. The preE-let-7g complexstructure described herein illustrates how the linker can adapt todifferent RNA substrates; Arg122 and Arg123 at the amino terminal end ofthe inter-domain linker stack against extra bases near the ds-ssjunction (FIG. 12B).

The GGAG tetranucleotide motif is well conserved among the members ofthe let-7 family within a particular species. In evolutionarily distantorganisms such as worms and fruit flies, however, other sequences (suchas GGUG or AUCA) are found in place of GGAG, perhaps due to co-evolutionof RNA and protein. Although not included in the crystal structure, thetwo nucleotides following GGAG are A and U in most let-7 sequences. Inthe context of full-length molecules, there may be more contacts betweenthe bulge near GGAG and CCHCx2. The importance of the GGAG motif hasbeen explored previously, by introducing a GGAG motif into an unrelatedRNA sequence, miR-16, to generate a chimeric pre-miRNA that has gainedaffinity for Lin28 (Heo et al., 2009). From the binding experiments andstructural data, the GGAG motif alone cannot confer robust binding withLin28, and shifting its position by a base or two relative to the CSDbinding site does not affect Lin28 binding significantly. In the case ofthe chimeric RNA with miR-16, its preE also coincidentally contains asequence similar to the preferred CSD binding site (UAAGAUUCU vs.NGNGAYNN), at the 5′ side of the GGAG motif, explaining why this chimeracan bind Lin28. The structural and biochemical data disclosed hereinthus provide a molecular explanation for Lin28 specificity, making itpossible to investigate further its role in let-7 biogenesis as well asits function in binding various mRNA targets (Jin et al., 2011,Peng etal., 2011,Qiu et al., 2010).

Implications for miRNA regulatory mechanisms: Although Drosha and Dicerare known to cut at opposite ends of the mature miRNA, there are stillmajor questions regarding how they recognize their target and how thecleavage can be regulated. The structures of Lin28:preE-let-7 complexesdisclosed herein combined with known structural data for Dicer haveallowed the inventor to discover how the Lin28 binding event itself caninhibit processing of pre-let-7 in at least two ways (FIG. 13D). First,Lin28 can act as a “wedge” to melt part of the double-stranded matureregion as it bends GGAG and situates itself in a particular conformationon one of the strands. As a result, Dicer can be unable to recognize itssubstrate properly. Second, given the location of CCHCx2 binding site,the volume of CCHCx2, and the location of its N-terminus from which theinterdomain linker would have to traverse to CSD, Lin28 is likely toclash with the Dicer dsRNA binding domains and also mask one of thecleavage sites.

The role of the preE in Drosha processing is less clear, especiallysince the Drosha cleavage site is at the opposite end of the matureregion from preE. Nonetheless, the direct association of Lin28 with thepreE shows that the observed effects of both the preE modifications andLin28 on Drosha activity are probably linked (Michlewski et al., 2008,Zeng, 2003, Zeng and Cullen, 2005, Zeng et al., 2005, Zhang and Zeng,2010). Other small RNA-binding proteins such as hnRNP-A1 and KSRP havebeen proposed to modify Drosha processing by binding to the preE region(Michlewski and Caceres, 2010, Michlewski et al., 2008). Rather thanbeing a mere by-product of miRNA processing, preE is clearly a criticalhandle for regulatory factors such as Lin28.

The mutagenesis studies disclosed herein indicate that the GGAG:CCHCx2region has an important functional role in regulating let-7, in additionto contributing to the specificity and tightness of complex formation.The in vitro binding results show that the observed strong effect ofmutations in the CCHCx2:let-7 interface cannot be attributed to theoverall affinity of the molecules alone. As the GGAG motif is closer tothe mature sequence, mutations that lead to lower occupancy at thissite—regardless of association of CSD with preE—can be more directlylinked to hindrance of processing enzymes. Moreover, the specificconformation of CCHCx2:GGAG induced by complex formation, as observed inour crystals, is probably important for recruiting downstream factor(s)such as TUTase. The critical role of Y140 of CCHCx2 in determining theRNA conformation is described in Results, and a uracil base (in GGAUmutant) would not be large enough to stack against Y140 efficiently inthe observed conformation. Transition mutations in GGAG sequence mightalso result in slightly different conformations, without greatlyreducing complex formation. Some of these mutations (to GAGG or AAGG)maintain their affinity for Lin28, but can obliterate uridylation byTUTase (Heo et al., 2009). That is, the CSD provides a larger contactand contributes more strongly than CCHxCx2 to let-7 affinity, but thelatter domain has additional effector functions.

The structures of the three Lin28:preE-let-7 complexes the inventorshave determined show a bipartite interaction of Lin28 with its let-7family partners (FIG. 7). The CSD inserts into the loop at one end ofthe central stem-loop structure in preE-let-7, and the CCHCx2 modulerecognizes a GGAG motif at the other end. The linker between CSD andCCHCx2 is flexible, to accommodate variable sequences and lengths amongLin28-regulated let-7 family members without compromising affinity orspecificity. This molecular organization explains several conservedfeatures of preE-let-7s: first, a minimum loop length of 9-nucleotides,with a preferred sequence of NGNGAYNNN; second, a stem-like structurethat closes the loop into a circle; and third, a GGAG motif close to the3′ end of the preE. The model provided by our crystal structuresprovides a mechanistic explanation for the inhibitory effect of Lin28 onmiRNA processing by Dicer; it further suggests that the CCHCx2:GGAG partof the complex directly influences downstream factor(s) important forlet-7 regulation. These structural details will be useful for developingtherapies that target the Lin28:pre-let-7 complex and its effects onlet-7 processing.

Thus, presented herein are high-resolution crystal structures of mouseLin28a in complex with three preE constructs of let-7d, let-7f-1, andlet-7g. These structures provide a direct view of a protein interactingwith the terminal loop region of a miRNA. The inventors have discoveredsequence-specific interactions between Lin28 and let-7 precursors thatgive direct structural evidence for the role of preEs in miRNAregulation. The Lin28 CSD and the CCHC “zinc knuckles” make extensivecontacts with the preE elements in two distinct regions. Describedherein also are NMR studies and biochemical assays showing that thelinker between the CSD and CCHCx2 regions introduces flexibility toaccommodate variable preE sequences and lengths while preserving thejoint contribution of the two interaction sites to overall affinity. Thedata show that both the terminal and linker regions outside of thefolded domains are not essential for blocking let-7 in vivo. Studieswith mutagenesis of preE fragments and full-length pre-miRNA show thespecificity of Lin28 and how Lin28 recognizes other let-7s. Complexformation induces in both Lin28 and preE-let-7 a specific conformationthat can affect recognition by downstream factors such as Drosha, Dicerand TUTase, and changes in the CCHCx2 region are particularlydetrimental to Lin28 activity in vivo.

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All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

LENGTHY TABLES

The patent application contains three (3) lengthy Tables; Table 1, Table2, and Table 3. A copy of the Tables (Table 1, Table 2, and Table 3) areavailable in electronic form from the USPTO web site. An electronic copyof the table will also be available from the USPTO upon request andpayment of the fee set forth in 37 CFR 1.19(b)(3).

1. (canceled)
 2. A polypeptide comprising amino acids 31-187 of awild-type Lin28 polypeptide and wherein the polypeptide comprises adeletion in the amino acid sequence connecting the CSD and the firstCCHC domain of the wild-type Lin28 polypeptide.
 3. The polypeptide ofclaim 2, wherein the polypeptide comprises a deletion of at least fiveamino acids in the amino acid sequence connecting the CSD and the firstCCHC domain of the wild-type Lin28 polypeptide.
 4. The polypeptide ofclaim 2, wherein the comprises a deletion between amino acid positions121 to 138 of the wild-type Lin28 polypeptide.
 5. The polypeptide ofclaim 4, wherein the polypeptide comprises a deletion of at least fiveamino acids between positions 121 to 138 of the wild-type Lin28polypeptide.
 6. The polypeptide of claim 5, wherein the polypeptidecomprises a deletion of amino acids 127 to 135 of the wild-type Lin28polypeptide. (New) The polypeptide of claim 5, wherein the polypeptidecomprises a deletion of amino acids 121 to 135 of the wild-type Lin28polypeptide.
 8. The polypeptide of claim 2, wherein the polypeptide isless than 200 amino acids in length.
 9. The polypeptide of claim 2,wherein the polypeptide comprises at least one modification.
 10. Thepolypeptide of claim 9, wherein the modification is selected from thegroup consisting of a non-natural amino acid, a D-amino acid, a β aminoacid, a chemically modified amino acid, a modified amide linkage, a tagamino acid sequence, and any combinations thereof.
 11. The polypeptideof claim 2, wherein the polypeptide is complexed with a divalent cation.12. The polypeptide of claim 11, wherein the divalent cation is selectedfrom the group consisting of zinc, cobalt, nickel, cadmium, magnesium,and manganese cations.
 13. The polypeptide of claim 12, wherein thedivalent cation is Zn²⁺.
 14. The polypeptide of claim 2, wherein thepolypeptide is complexed with a nucleic acid.
 15. The polypeptide ofclaim 14, wherein the nucleic acid comprises the nucleotide sequence5′-N¹N²N³N⁴N⁵N⁶N⁷N⁸N⁹-X-GGAG-3′, wherein N², N⁴, and N⁵ areindependently a purine; N⁶ is a pyrimidine; N¹, N³, N⁷, N⁸, and N⁹ areindependently any nucleotide; and X is a nucleotide sequence of from 0to 100 nucleotides.
 16. The polypeptide of claim 2, wherein thewild-type Lin28 polypeptide is a Lin28A or Lin28B polypeptide.
 17. Thepolypeptide of claim 2, wherein the wild-type Lin28 polypeptide is ahuman Lin28 polypetide.
 18. A method of inhibiting miRNA processing of apri-miRNA to a mature miRNA in a cell, the method comprising contactinga cell with a polypeptide of claim
 2. 19. A screening assay fordetermining inhibitor of Lin28 activity, the method comprisingcontacting a polypeptide claim 2 with a test compound and selecting thecompound that increases level of mature let-7 miRNA relative to acontrol.
 20. The method of claim 19, wherein the test compound has amolecular weight of less than 5000 Daltons (5 kD).
 21. The method ofclaim 19, wherein the method is a high-throughput screening method.